Cell Membranes

FUNCTIONS  1. Compartmentalization Membranes enclose compartments --- plasma membrane encloses entire cell----nuclear and cytoplasmic membranes enclose various internal cellular spaces in which specialized activities take place.

 2. Provides a selectively permeable membrane barrier Movement of molecules either into or out of cell is regulated . Only very small uncharged molecules can readily diffuse i.e.. O2 and CO2 (Fig. 1).

 3. Provides for the transportation of solutes The membrane contains the machinery for physically transporting substances from one side of the membrane to another --- even against a concentration gradient (Fig. 1).

 4. Ability to respond to external signals Receptors on surface have a particular ligand. When ligand binds to the receptor the signal must be transduced to the nucleus. Signal Transduction  (Fig. 1) Hormones, growth factors, neurotransmitters can all serve as ligands.

 5. Provides for intercellular and intracellular interactions

cell-cell adhesion cell-ECM adhesion linkers add strength and shape to membrane and localize other proteins  (Fig. 1).

 6. Serves as a site for biochemical activities Organizes cellular activities by the association of multi-enzyme complexes i.e. electron transport chains.

7. Provides for energy transduction Transduction refers to the conversion of one type of energy into a different form of energy. Ex: the energy of sunlight is absorbed by membrane-bound photosynthetic pigments and converted to chemical energy contained in carbohydrates.

 Conversely- the chemical energy in carbohydrates and fats can be converted to ATP.

(Text: p. 77-84 & 467-476)

 STRUCTURAL PROPERTIES OF CELL MEMBRANES  Formation of cell membranes is based upon the properties of lipids.

 All are bi-layers of phospholipids with associated proteins. As previously mentioned, phospholipids are amphipathic meaning that they possess both a hydrophobic and hydrophilic end (Fig. 2).

 

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Polar head groups are in contact with water while fatty acid tails aggregate in the interior of the membrane.

 The four major phospholipids found in cell membranes:

phosphatidyl choline (Fig. 2) phosphatidyl ethanolamine (Fig. 2) phosphatidyl serine (Fig. 2) sphingomyelin - a non-glycerol phospholipid (Fig. 3)

  Various glycolipids are also found in the outer leaflet of the cell membrane.

 Cholesterol is another important constituent of the animal cell membrane (Fig. 4 ) .

 Lipid composition differs in the different types of cells and in different types of organisms. The average eukaryotic plasma membrane - ~50% of mass is lipid and 50% protein.

 The lipid bilayer behaves as a fluid.

 FLUID MOSAIC MODEL The fluid mosaic model was first proposed by Singer and Nicolson . Lipids and proteins can readily move laterally and can also undergo rotation (Fig.1). The degree of membrane fluidity is determined by temperature and lipid composition.

 Lipids with shorter fatty acid chains are less rigid and remain fluid at lower temperatures. This is because interactions between shorter chains is weaker than for longer chains.

 Lipids containing unsaturated fatty acids increase membrane fluidity The = bonds introduce kinks, preventing tight packing of the fatty acids (Fig. 2).

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 Cholesterol (Fig. 4 ) with its hydrocarbon ring structure plays a distinct role in determining membrane fluidity. Polar hydroxyl group positions close to the phosphate head group. Rigid rings interact with regions of fatty acid chain adjacent to phospholipid head groups. This interaction decreases the mobility of the outer portions of the fatty acid chains, making this region of the membrane more rigid, even at higher temperatures.

 On the other hand... Insertion of cholesterol interferes with interactions between fatty acids, thereby maintaining fluidity at lower temperatures.

 Cholesterol is not present in bacteria or plant cells.

 Plant cell membranes do contain sterols which function in a manner similar to cholesterol.

 In the fluid mosaic model of the membrane ----there are membrane proteins inserted into the lipid bilayer. The lipids provide the basic structure, but proteins carry out the specific functions of the different types of membranes (Fig. 1 ) .

 

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Cell Surface(Cooper, 1997 p. ) 

MEMBRANE PROTEINS

Integral membrane proteins

Amphipathic Properties Also referred to as transmembrane proteins. Commonly, integral membrane proteins have membrane spanning domains which are alpha helical. May be 1 , 2, 7 or more membrane spanning alpha helical domains. The alpha helix neutralizes the polar character of the peptide bonds. The hydrophobic side chains assoc. with these amino acids interact with the fatty acid chains of membrane lipids.

 Most transmembrane proteins are also glycosylated [have carbohydrate groups attached]

 Peripheral membrane proteins

Not embedded in the bilayer but indirectly associated with the membrane through interactions with integral membrane proteins or by weak electrostatic interactions with the hydrophilic head groups of membrane lipids. Located extracellular or associated with the cytoplasmic surface of the bilayer.

 Lipid-anchored proteins

Located outside the lipid bilayer, but covalently linked to a lipid molecule that is situated within the bilayer. An increasingly large # of proteins have been found to be linked by a short oligosaccharide to a molecule of glycophosphatidylinositol (GPI) that is embedded in the outer leaflet of the lipid bilayer. These proteins are released when membrane is treated with enzymes that (Phospholipases) that specifically recognized and cleaved inositol-containing phospholipids.

 Another group of proteins are actually present on the cytoplasmic side of the membrane and are anchored by long hydrocarbon chains embedded in the inner leaflet of the lipid bilayer.

 THE GLYCOCALYX

The extracellular portion of the plasma membrane proteins are generally glycosylated. Likewise, the carbohydrate portions of glycolipids are exposed on the outer face of the plasma membrane. Consequently, the glycocalyx, is formed by the oligosaccharides of glycolipids and transmembrane glycoproteins.

 Role: Protection of cell surface Markers for cell-cell interactions

TRANSPORT ACROSS CELL MEMBRANES

Membranes are selectively permeable --- small, uncharged molecules can diffuse freely through the phospholipid bilayer. O2, CO2

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Also some small polar molecules H2O, ethanol and some small relatively hydrophobic molecules such as benzene.

Passive diffusion

Molecule simply dissolves in the phospholipid bilayer, diffuses across it, and then dissolves in the aqueous solution at the other side of the membrane. No membrane proteins involved in process. The net movement of molecules is simply according to the concentration gradient, with molecules moving from an area of higher concentration to an area of lower concentration.

 Water is actually a rather special case - readily diffusable according to concentration gradient. Process is known as osmosis.

 Larger polar molecules such as glucose cannot cross by passive diffusion.

 Charged ions also cannot [Na+, Ca++, K+, Cl-], even H+ cannot.

 Facilitated Diffusion

Like passive diffusion, involves the movement of molecules in a net direction determined by concentration gradient---but with the assistance of specialized proteins.

 Channel Proteins Proteins which form open pores allowing for free passage of any molecule of appropriate size and charge by free diffusion. Form a passage through the lipid bilayer, allowing polar or charged molecules to cross without interacting with the hydrophobic fatty acid chains of the phospholipids. ION CHANNELS-specific example of a channel protein. not permanently open [GATED] very wide variety of ion channels All are integral membrane proteins that surround an aqueous pore. Bidirectional flow of ions based upon the electrochemical concentration gradient Most channel proteins are said to be gated meaning that they can exist in open or closed conformation.

 Carrier Proteins [Transporter Proteins] Selectively bind and transport specific small molecules such as glucose. The molecules bind and the protein undergoes a conformational change that allows specific molecules to pass through.

 facilitated diffusion of sugars, amino acids, and nucleosides.

 Active transport

Carrier proteins also provide a mechanism through which the energy changes associated with transporting molecules can be coupled to the use or production of other forms of metabolic energy.

 Molecules can be transported against a concentration gradient if the transport is coupled to ATP hydrolysis, the absorption of light, the transport of electrons, or the flow of other substances down a gradient - as a source of energy.

 Typically the K+ concentration inside a mammalian cell is about 100 mM, while that outside the cell is only 5mM. Diffusion of potassium out of the cell is favored. Sodium ions and Calcium ions

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have the opposite concentration gradient. Such gradients are maintained by active transport. ION PUMPS

 Depends on integral membrane proteins that are capable of selectively binding a particular solute and moving that substance across the membrane- driven by changes in the protein's conformation.

 

The Endocytic Pathway

Uptake of macromolecules and particles is done by ENDOCYTOSIS. Pinocytosis effectively internalizes portions of the cell membrane, any proteins or receptors on the membrane, and any ligands attached to those receptors. Fate of these receptors and their ligands varies after endocytosis. When soluble Ig binds to antigen, both receptor and ligand are directed to the lysosmes. The other possible route is that vesicles may be transported to another region of the membrane where the ligand is released.

 One form of pinocytosis involves the use of coated pits [receptor-mediated endocytosis]. Ligand binds to receptor. Complex travels laterally through the membrane to a coated pit region. Complexes are retained and concentrated in these pits. Clathrin is a protein whose subunits form the surface of the pit. Pit then invaginates and eventually forms a vesicle known as a clathrin coated vesicle. The clathrin coated vesicle first fused with vesicles known as early endosomes. Fusion with the early endosome brings the pH of the clathrin coated vesicle down to between pH 6 and 6.2. This shift to an acidic pH allows the clathrin and the receptor to be transported back to the surface. Late endosomes then fuse with the vesicle, further lowering the pH to between 5.5-6.0. Finally, lysosomes fuse bringing the pH down to about 5.0. Lysosomes also release a battery of hydrolytic enzymes that digest the material remaining in the vesicle. The other major form of endocytosis is termed: Phagocytosis

 Phagocytosis can only be carried out by phagocytic cells such as macrophages and neutrophils. Phagocytosis involves the internalization of particles such as bacteria, protozoa, etc. In the process of phagocytosis, the particle first binds to the phagocytic cell, then the cell sends out extensions of the cyotplasm known as pseudopodia which surround the particle. The formation of psuedopodia is dependent upon the polymerization of the cytoskeletal protein, actin. The internalized particle is now enclosed in a membrane-bound structure known as a phagosome. Lysosmes subsequently fuse with the phagosome forming a phagoslysosome. The hydrolytic enzymes released by the lysosome function in the digestion of the internalized particle.

 Exocytosis

Material enclosed in a cell vacuole is passed to the extracellular fluid by fusion of the vacuole with the plasma membrane. Secretory process and a mechanisim of replenishing lipids and proteins of plasma membrane.  

Eukaryotic Cell StructurePreface: Prokaryotic Cells  = "before the nucleus" Some prokaryotic cells do have a nucleoid region, in which DNA is concentrated, but the DNA is

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not enclosed in a separate membrane. In addition, prokaryotic cells lack membrane-bound organelles.

Eukaryotic Cell Structure.

 All eukaryotic cells possess membrane-bound organelles that divide them into compartments and each represents a structure with a distinct function.

 In addition, eukaryotic cells possess a distinct nucleus surrounded by two membranes that constitute the nuclear membrane.

 Cytoplasm - region between the nucleus of the cell and the plasma membrane. Term includes the cytosol and all of the organelles except for the nucleus.

 Cytosol - refers to the fluid component of the cytoplasm.

Nucleoplasm- corresponding material within the nucleus.

 Cells have to grow, replace worn-out parts, and repair themselves. They must synthesize the proteins and other complex molecules of which they are made.

ENDOPLASMIC RETICULUM

Extensive maze of branching membranes that extends throughout the cytoplasm. Evidence suggests that the ER is continuous with the plasma membrane and with the outer membrane surrounding the nucleus. (Drawing) [Intramembrane network]

 Therefore, the membranes of the ER divide the cytoplasm up into interconnected compartments in which different types of reactions take place.

The ER plays an important role in the synthesis of proteins and some lipids. Expanded regions of the ER may serve as storage areas. The ER also functions as a system for transporting materials from one part of the cell to another.

 Smooth ER

 The smooth ER is more tubular and its outer membrane surfaces have a smooth appearance. It is the primary site of phospholipid, steroid, and fatty acid metabolism.

 The smooth ER also contains enzymes that detoxify harmful chemicals, breaking them down into water-soluble substances that can be excreted.

 Rough ER

 The Rough ER has a more granular appearance under EM due to the presence of ribosomes.

 Ribosomes of course serve as the sites of protein synthesis and can be found assoc. with the ER or free in the cytoplasm. RER is especially abundant in cells that synthesize proteins destined for exocytosis.

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 GOLGI COMPLEX

Factory for packaging proteins Consists of stacks of flattened membranous sacs that may be distended in certain regions because they are filled with cell products.

The golgi complex processes, sorts, modifies, and packages proteins. Proteins that pass through the golgi are:

secreted from the cell plasma membrane proteins and proteins routed to other intracellular organelles pass through the golgi. Such proteins are synthesized on ribosomes associated with the RER. From the ER they are transported to the Golgi complex in small membrane-bound vesicles formed from the ER membrane.

 These vesicles then fuse with the membranes of the Golgi complex. The proteins then pass through the separate layers of the Golgi complex moving by way of membrane transport vesicles.

 During their passage- proteins are modified in different ways Carbohydrates are often added here or previously added carbohydrates are modified.

 In some cases, the carbohydrates and other modifications act as "sorting signals" allowing the GOLGI to route the protein to different parts of the cell. The GOLGI of plant cells also produces some of the extracellular polysaccharides used in components of the cell wall.

The Golgi Complex has a distinct polarity. The cis face of the golgi {closest to the nucleus} is the site where membrane-bound vesicles from the ER first fuse. The golgi stack refers to the middle portion of the golgi. The trans face of the golgi refers to the portion of the golgi closest to the plasma membrane and serves as the site where membrane-bound vesicles bud and then exit the golgi complex.

 The Golgi also performs important functions in non-secreting cells by packaging intracellular digestive enzymes in the little organelles known as lysosomes.

 LYSOSOMES

Small membrane-bound vesicles that contain digestive (hydrolytic enzymes). The vesicles are released from the GOLGI COMPLEX and are dispersed throughout the cytoplasm. Over 50 different enzymes present.

Lysosomes play an important role in the digestion of internalized particles (phagocytosis) and macromolecules (pinocytosis/receptor-mediated endocytosis).

 Another important function of lysosomes is the digestion and break down of old cellular components, which otherwise tend to accumulate and interfere with proper cell function. The lysosome membrane itself is able to resist the digestive action of its own enzymes.

 In addition, when a cell dies, the lysosome membrane breaks down, releasing digestive enzymes into the cytoplasm, where they break down the cell itself.

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Some forms of tissue damage as well as the aging process may be related to "leaky lysosomes".

Whether or not lysosomes are present in plant cells is open to debate.  

 VACUOLES May carry out many of the function of the lysosome in the plant cell and algal cell. Contains acid hydrolases and the pH of the vacuole is maintained at a low value.

 As much as 90% of the volume of a plant cell may be occupied by a large central vacuole containing water, stored food, salts, pigments, and wastes. Plants lack systems for disposing of metabolic waste products that are toxic to the cells. Such waste products often aggregate and form small crystals inside the vacuole. The vacuole plays an important function as a storage compartment. Compounds noxious to predators or various compounds used in the plant defense against pathogens may be stored here.

 Vacuoles are also involved in plant cell enlargement and in maintaining cell rigidity. Vacuoles are also present in several different types of animal cells and in many protozoa. Protozoa often have food or digestion vacuoles which fuse with lysosomes so that the food can be digested. Many also have contractile vacuoles which function to remove excess water from the cell.

 MICROBODIES

Membrane-bound organelles that contain enzymes that regulate many different metabolic reactions. One type of microbody, the peroxisome, regulates the converstion of fats to carbohydrates. During the breakdown of fats, hydrogen peroxide is produced. Peroxisomes contain enzymes (including Catalase) that split hydrogen peroxide into water and oxygen, making it harmless. Proteins found in the peroxisome are synthesized in the cytoplasm of the cell and then transported to the peroxisome.

 Peroxisomes often contain a dense crystalline core consisting of one of the oxidative enzymes. Peroximsomes in the liver and kidney cells may be important in detoxifying certain compounds such as ethanol in alchoholic beverages. Peroxisomes occur in both plant and animal cells.

 Glyoxysome Abundant in the seeds of certain plants. Its enzymes convert stored fats to sugars. These sugars are used as an energy source and as a component for making needed compounds during the germination of seeds. Contains catalase and the enzymes for fatty acid oxidation as do peroxisomes.

MITOCHONDRIA

Typical mitochondrion is sausage-shaped and similar in size to a bacterium. Sometimes they can assume a more spherical shape and sometimes even a long thread-like and even branching shape. In other words, the structure of the mitochondrion is dynamic, changing. (Drawing) (Electron Micrograph)

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 The mitochondria play a central role in making chemical energy available to the cell. [ATP SYNTHESIS] Cells which require and expend a lot of energy typically have a lot of mitochondria (Muscle cells).

 The internal structure of the mitochondrion can be diagrammed schematically. See diagrams in text.

 Each mitochondrion contains an outer membrane and a complex inner membrane system. The outer membrane completely encloses the mitochondrion, serving as its outer boundary. The inner membrane lies beneath the outer membrane but has deep folds or invaginations called cristae. In some cells the cristae are wide sheets that cut across the entire diameter of the mitochondrion. In most plant cells, the cristae have more of a tubular shape. These infoldings greatly increase the amount of surface available to house machinery needed for aerobic respiration.

 Inner aqueous compartment is called the matrix.

 Between the outer and inner membrane is the inter membrane space.

 AT least 60 different types of polypeptides are found in the inner membrane of the mitochondrion. Mass protein/lipid ratio is 3:1 by weight which translates into 1 protein for every 15 phospholipids. The inner membrane is devoid of cholesterol and rich in an unusualy phospholipid- cardiolipin. What I have just described to you is VERY similar to the structure of the bacterial membrane.

 The outer membrane contains PORINs which are integral membrane proteins that form large, non-selective membrane channels (also found on outer membrane of certain bacterial cells as part of the cell wall). Outer membrane of mitochondrion is especially permeable and allows molecules up to 10,000 Daltons to pass freely into the intermembrane space. The inner membrane is highly impermeable; virtually all molecules and ions require special transporters. In addition to containing a variety of transport systems, the inner mitochondrial membrane contains most of the enzymes required for the synthesis of ATP.

 Matrix The matrix contains a variety of enzymes, ribosomes, and molecules of double-stranded DNA (usually circular). This nonchromosomal DNA is important because it encodes a small number of mitochondrial polypeptides that are integrated into the inner mitochondrial membrane together with polypeptides encoded by genes residing within the nucleus and imported from their site of synthesis in the cytosol.

 Observations of living cells show that mitochondria are dynamic organelles that change their shape, move from place to place within the cytoplasm, and undergo branching. Mitochondria arise by fission from pre-existing mitochondria.

Mitochondria are often described as miniature power plants. They extract energy from organic materials and store it in the form of electrical energy.

 Following glycolysis, [which occurs in the cytoplasm of the cell], the pyruvate is transported across the inner mitochondrial membrane and into the matrix where it is further broken down in the tricarboxylic acid cycle (Krebs cycle). All but one of the enzymes used in this cycle are in the matrix- one is positioned in inner membrane. Krebs original description of the cycle was rejected in the 1930s by NATURE!!

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 Final phase of aerobic cellular respiration is OXIDATIVE PHOSPHORYLATION which results in the largest net gain of ATP. ET [electron transport] chain is located in the inner mitochondrial membrane.

 CHLOROPLASTS Chloroplasts are the organelles responsible for photosynthesis and function to generate metabolic energy. Structural characteristics:

o They have a double membrane around the outside and have an additional thylakoid membrane that can for stacks of disks called grana.  (Diagram)

o They have their own genetic material and perform protein synthesis o They synthesize their own fatty acids, amino acids, and lipid components o They reduce nitrite (NO2

-) to ammonia (NH3).

Chloroplasts have three distince internal compartments

1. The space between the two outer membranes 2. The stroma within the double unit membrane but outside the thylakoids

3. The space within the thylakoids

Like mitochondria the outer membrane contains porins and is permeable to small molecules while the inner membrane is very restrictive.

Unlike the mitochondria, the electron transport and energy production of chloroplast is located in the thylakoids instead of the inner membrane.  In chloroplasts hydrogen ions are pumped into the thylakoid during photosynthesis and ATP is generated as these ions pass out into the stoma.

Like mitochondria, chloroplasts contain their own genetic material, perhaps reflecting their evolutionary origins as photosynthetic bacteria.  The chloroplast genome is much larger, 120 - 160 kbp, and codes for about 120 genes.

About 90% of choroplast proteins are encoded by nuclear genes and are transported from the cytoplasm of the cell into the chloroplast via signal peptides.

Other Plastids

Chromoplasts lack chlorophyll but contain carotenoids and other pigments and are responsible for the color of flowers and fruits.

Leucoplasts are nonpigmented plasteds which sore energy sources in nonphotosynthetic tissue.

o Amyloplasts are Leucoplasts that store starch. o Elaioplasts are leucoplasts that store lipids.

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How do proteins translocate into the lumen of  the rough endoplasmic reticulum?

What is different about the protein that is destined for the rough endoplasmic reticulum?

[Note: This section describes work that led to a Nobel Prize in Medicine and Physiology to Dr. Gunter Blobel.  For more information about Dr. Blobel's work and the pioneering discoveries, click: http://www.nobel.se/medicine/laureates/1999/ ]

The major difference is the fact that it has a hydrophobic signal sequence.  This simplified cartoon shows that this is the first part of the protein produced.  After the signal sequence is completed, protein synthesis is further inhibited.  This is to allow the interaction of the signal sequence with a complex on the rough endoplasmic reticulum.  In the above cartoon, note that the signal peptide is allowed to enter and essentially guide the protein into the lumen of the rough endoplasmic reticulum.  Once the signal sequence is detected, protein synthesis resumes and the rest of the protein is inserted in the lumen.  Note that a signal peptidase near the inner surface of the membrane works to cleave the signal sequence from the growing peptide.

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[The text reading for this discussion is Alberts et al, Molecular Biology of the Cell, third edition, Garland Publishing, 1994, pp 577-588 (Chapter 12) and pp 599-616. All of the figures in these web pages are linked to a page listing the citation from which the figure was taken.

Click on the figure to learn the citation. If there is no link, the figure came from our own collection ]

The complex is actually more complicated than the above. The cartoon to the left shows a view of the signal sequence binding and interaction

Note that the signal sequence is recognized by a Recognition Particle, or SRP.   This is then bound to a receptor.  This complex guides the protein through a channel like region.  It also consists of a docking site for the ribosome.

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Another cartoon view of this process shows the signal receptor peptide (SRP) that associates with the large subunit of the ribosome that allows binding to the receptor on the rough endoplasmic reticulum.

After the protein is synthesized, the ribosome dissociates into large and small subunits and the SRP also looses its attachment to the receptor.   

Current studies of ribosomal interactions with ER

Andrea Neuhof, M.M. Rolls, B. Jungnickel, K-U Kalies and T A Rapoport, of Signal Recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction. Molecular Biology of the Cell, 9: 103-115. 1997

Proteins destined for RER sorting make a signal sequence. As signal sequence elongates, it is bound by the signal recognition particle (SRP54)

(GTP dependent binding). SRP then binds to the SRP receptor (docking protein) on the ER membrane. At the same time, ribosomes bind to the RER translocation channel formed by the

Sec61p complex. This Sec61p complex is a major component of a protein-conducting channel which

also includes the “translocating chain-associating membrane protein” or TRAM. This conducts the protein into the RER sac.

Neuhof et al, 1997 have asked the following question: What stimulates the specificity of ribosomal docking?

Question was asked because ribosomes dock to the Sec61p complex even without the signal sequence. Some clues: 

As the signal sequence begins to appear (short) it can be removed from the ER membrane with high salt concentrations.

As the signal sequence elongates, ribosome binding is stronger and

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ribosome complex becomes insensitive to proteases and high salt. Neuhof's study hypothesized that the presence of the signal

peptide was crucial for specific binding. Study Design:

Added nontranslating ribosomes to compete with translating ribosomes in an in vitro

If Signal receptor protein (SRP) was absent, the nontranslating ribosomes bound to the Sec61p receptor on ER membranes.

Also, the nontranslating ribosomes competed with the translating ribosomes. Added SRP to bind to the signal peptide.

The translating ribosomes bound tightly and were not displaced. Then, nontranslating ribosomes failed to compete for the Sec61p receptor

sites.

Hence, SRP gives the translating ribosome a competitive edge once it starts translating the signal sequence.

 

  Return to Menu

 

You can see a better surface view in this cartoon.  The cartoon is from your text. It shows the

Ribosome sitting on the receptor next to the pore.  The signal peptide is noted in red. The remainder of the code is read as the ribosome moves along the mRNA. The fluidity of the membrane allows the ribosome to be docked at its receptor site and also move along the mRNA.  Each amino acid is added to the growing chain and the polypeptide gets longer. 

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This cartoon is also from your text.  It shows the system without the ribosome.  Here you get a better view of the pore through which the protein projects into the lumen and the signal sequence.

How are newly synthesized proteins inserted in the membrane?

M •

Type I: Signal sequence on amino terminus enters first and continues to elongate. Protein is threaded through the translocating channel (open area in rer membrane) until a hydrophobic stop sequence is reached. That hydrophobic stop sequence (seen as a hatched region in the protein) is then inserted in the membrane and forms the anchor for that protein. Signal is cleaved by protease inside the lumen.

•

 

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•

Type II: No cleavable signal sequence. These proteins have rather long hydrophobic regions that will be anchored in the membrane. Type II proteins are threaded into the lumen with the C terminus leading. Protein continues to be inserted until it reaches the hydrophobic stop signal sequence. 

•

 

•

Type III: Same as Type II, only the N terminus leads into the lumen.

 

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What regulates the orientation of Type II and III proteins?

•

Wahlberg, JM. And Spiess, M. Multiple determinants direct the orientation of signal anchor proteins: The topogenic role of the hydrophobic signal domain. J Cell Biology 137: 555-562.

• Tested charged amino acids and length of hydrophobic signal sequence.

The "positive inside rule" states that amino acid residues nearest the cytosolic side of the hydrophobic anchor sequence are more positive than those nearest the lumenal side.  So, whichever end has the least positive charges near the signal anchor patch would go into the ER lumen.  One can change the direction of translocation of a protein (reverse it) by mutating the protein and making more positively charged groups near the anchor patch of the other end. Below, the cartoon shows that this can be done to change a Type II protein (COOH end enters ER lumen) to a Type III (which has its amino terminal entering the lumen).

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Washburn and Speiss (JCB 137: 555-562, 1997) also tested the length of the hydrophobic signal anchor sequence. The following cartoon shows that a longer hydrophobic anchor sequence (seen as the portion running through the membrane) promotes entry with the amino terminal leading into the lumen.

 

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Proteins destined for insertion into membranes, such as ion channels or receptors have mRNA codes for start and stop sequences that allow multiple passes through the membrane.  Signalling sequences (patches) can be formed as described in the above cartoon. It shows the insertion of a double pass transmembrane protein with the loop inside the rough endoplasmic reticulum. The red signal patch has the + charges near the cytosol and starts the insertion process.  This continues until the hydrophobic stop signal patch is reached.  That anchors the second membrane passage.  Note, that both C and N terminal portions are in the cytosol  Thus, if this protein is destined for the plasma membrane, that loop in the ER lumen will eventually project outside the cell

Membrane proteins that pass through the membrane multiple times (called "multipass transmembrane proteins) have multiple start and stop signals. They are aligned with the hydrophilic and hydrophobic portions of the lipid bilayer as described in the lecture on membranes. This is shown in the following cartoon

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How do proteins translocate into the lumen of  the rough endoplasmic reticulum?

What is different about the protein that is destined for the rough endoplasmic reticulum?

[Note: This section describes work that led to a Nobel Prize in Medicine and Physiology to Dr. Gunter Blobel.  For more information about Dr. Blobel's work and the pioneering discoveries, click: http://www.nobel.se/medicine/laureates/1999/ ]

21

The major difference is the fact that it has a hydrophobic signal sequence.  This simplified cartoon shows that this is the first part of the protein produced.  After the signal sequence is completed, protein synthesis is further inhibited.  This is to allow the interaction of the signal sequence with a complex on the rough endoplasmic reticulum.  In the above cartoon, note that the signal peptide is allowed to enter and essentially guide the protein into the lumen of the rough endoplasmic reticulum.  Once the signal sequence is detected, protein synthesis resumes and the rest of the protein is inserted in the lumen.  Note that a signal peptidase near the inner surface of the membrane works to cleave the signal sequence from the growing peptide.

[The text reading for this discussion is Alberts et al, Molecular Biology of the Cell, third edition, Garland Publishing, 1994, pp 577-588 (Chapter 12) and pp 599-616. All of the figures in these web pages are linked to a page listing the citation from which the figure was taken.

Click on the figure to learn the citation. If there is no link, the figure came from our own collection ]

The complex is actually more complicated than the above. The cartoon to the left shows a view of the signal

22

sequence binding and interaction

Note that the signal sequence is recognized by a Recognition Particle, or SRP.   This is then bound to a receptor.  This complex guides the protein through a channel like region.  It also consists of a docking site for the ribosome.

Another cartoon view of this process shows the signal receptor peptide (SRP) that associates with the large subunit of the ribosome that allows binding to the receptor on the rough endoplasmic reticulum.

After the protein is synthesized, the ribosome dissociates into large and small subunits and the SRP also looses its attachment to the receptor.   

Current studies of ribosomal interactions with ER

Andrea Neuhof, M.M. Rolls, B. Jungnickel, K-U Kalies and T A Rapoport, of Signal Recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction. Molecular Biology of the Cell, 9: 103-115. 1997

Proteins destined for RER sorting make a signal sequence. As signal sequence elongates, it is bound by the signal recognition particle (SRP54)

(GTP dependent binding). SRP then binds to the SRP receptor (docking protein) on the ER membrane. At the same time, ribosomes bind to the RER translocation channel formed by the

Sec61p complex. This Sec61p complex is a major component of a protein-conducting channel which

also includes the “translocating chain-associating membrane protein” or TRAM. This

23

conducts the protein into the RER sac.

Neuhof et al, 1997 have asked the following question: What stimulates the specificity of ribosomal docking?

Question was asked because ribosomes dock to the Sec61p complex even without the signal sequence. Some clues:

As the signal sequence begins to appear (short)ER membrane with high salt concentrations.

As the signal sequence elongates, ribosome binding is stronger and ribosome complex becomes insensitive to proteases and high salt.

Neuhof's study hypothesized that the presence of the signal peptide was crucial for specific binding. Study Design:

Added nontranslating ribosomes to compete with translating ribosomes in an in vitro

If Signal receptor protein (SRP) was absent, the nontranslating ribosomes bound to the Sec61p receptor on ER membranes.

Also, the nontranslating ribosomes competed with the translating ribosomes. Added SRP to bind to the signal peptide.

The translating ribosomes bound tightly and were not displaced. Then, nontranslating ribosomes failed to compete for the Sec61p receptor

sites.

Hence, SRP gives the translating ribosome a competitive edge once it starts translating the signal sequence.

 

  Return to Menu

 

You can see a better surface view in this cartoon.  The cartoon is from your text. It shows the

24

Ribosome sitting on the receptor next to the pore.  The signal peptide is noted in red. The remainder of the code is read as the ribosome moves along the mRNA. The fluidity of the membrane allows the ribosome to be docked at its receptor site and also move along the mRNA.  Each amino acid is added to the growing chain and the polypeptide gets longer. 

This cartoon is also from your text.  It shows the system without the ribosome.  Here you get a better view of the pore through which the protein projects into the lumen and the signal sequence.

How are newly synthesized proteins inserted in the membrane?

M •

Type I: Signal sequence on amino terminus enters first and continues to elongate. Protein is threaded through the translocating channel (open area in rer membrane) until a hydrophobic stop sequence is reached. That hydrophobic stop sequence (seen as a hatched region in the protein) is then inserted in the membrane and forms the anchor for that protein. Signal is cleaved by protease inside the lumen.

•

 

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Type II: No cleavable signal sequence. These proteins have rather long hydrophobic regions that will be anchored in the membrane. Type II proteins are threaded into the lumen with the C terminus leading. Protein continues to be inserted until it reaches the hydrophobic stop signal sequence. 

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Type III: Same as Type II, only the N terminus leads into the lumen.

 

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What regulates the orientation of Type II and III proteins?

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Wahlberg, JM. And Spiess, M. Multiple determinants direct the orientation of signal anchor proteins: The topogenic role of the hydrophobic signal domain. J Cell Biology 137: 555-562.

• Tested charged amino acids and length of hydrophobic signal sequence.

The "positive inside rule" states that amino acid residues nearest the cytosolic side of the hydrophobic anchor sequence are more positive than those nearest the lumenal side.  So, whichever end has the least positive charges near the signal anchor patch would go into the ER lumen.  One can change the direction of translocation of a protein (reverse it) by mutating the protein and making more positively charged groups near the anchor patch of the other end. Below, the cartoon shows that this can be done to change a Type II protein (COOH end enters ER lumen) to a Type III (which has its amino terminal entering the lumen).

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Washburn and Speiss (JCB 137: 555-562, 1997) also tested the length of the hydrophobic signal anchor sequence. The following cartoon shows that a longer hydrophobic anchor sequence (seen as the portion running through the membrane) promotes entry with the amino terminal leading into the lumen.

 

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Proteins destined for insertion into membranes, such as ion channels or receptors have mRNA codes for start and stop sequences that allow multiple passes through the membrane.  Signalling sequences (patches) can be formed as described in the above cartoon. It shows the insertion of a double pass transmembrane protein with the loop inside the rough endoplasmic reticulum. The red signal patch has the + charges near the cytosol and starts the insertion process.  This continues until the hydrophobic stop signal patch is reached.  That anchors the second membrane passage.  Note, that both C and N terminal portions are in the cytosol  Thus, if this protein is destined for the plasma membrane, that loop in the ER lumen will eventually project outside the cell

Membrane proteins that pass through the membrane multiple times (called "multipass transmembrane proteins) have multiple start and stop signals. They are aligned with the hydrophilic and hydrophobic portions of the lipid bilayer as described in the lecture on membranes. This is shown in the following cartoon

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Golgi Complex: Structure and Function

How do proteins move to the Golgi Complex?Bannykh, S and Balch WE Membrane Dynamics at the Endoplasmic Reticulum-Golgi Interface. J Cell Biol 138: 1-4 (1997)

Tier I shows budding from ER that is arranged facing a central zone at one end of the Golgi complex.  These buds become vesicles and are coated with COPII protein coats.  

Tier II The ER faces a central zone called a vesicular-tubular cluster (VTC). After they lose their COPII coat, they merge with the VTC's carrying the soluble and membrane proteins to the Golgi complex. 

Tier III illustrates the entire complex which is unique in the cytoplasm.  It is termed the 'export complex' and contains unique proteins that suggest it is specialized for information flow to and from ER and the Golgi complex.

The above drawing shows an actual interface between the ER and the Golgi complex.  The "Export complex" is seen at the top of the drawing.vesicle are moving to contribute to the cis-Golgi network of vesicles and

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cisternae.

The movement of these special transport vesicles is an energy requiring process. If one blocks production of ATP, the transport will not happen. This drawing shows how the rough endoplasmic reticulum forms vesicles (without ribosomes attached) that carry the newly synthesized proteins to the Golgi complex. 

The inside of the vesicle becomes continuous with the inside of the Golgi cisternae, so that protein groups pointing towards the inside, could eventually be directed to face the outside of the cell. 

Carbohydrate groups are attached and any subunits may be joined in these cisternae. The protein is then passed to the final region of the Golgi called the "trans face". There it is placed in vacuoles that bud from this region of the Golgi complex. These may be a certain size or density, characteristic of the cell itself. The vacuoles continue to condense the proteins and the final mature secretory granule is then moved to the membrane for secretion.

This electron micrograph illustrates a Golgi Complex. It is curved with its Trans face pointing away from the nucleus toward the cell periphery. The numerous vesicles in the area are transporting the proteins to and from cisternae.

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 Transport of material in and out of the Golgi complex involves budding and fusion of vesicles. This cartoon shows that the membranes of each join and align themselves during the process so that the inside face remains in the lumen and the outside face remains towards the cytoplasm.

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What types of secretion are controlled by the Golgi complex?

The Golgi complex controls trafficking of different types of proteins. Some are destined for secretion. Others are destined for the extracellular matrix. Finally, other proteins, such as lysosomal enzymes, may need to be sorted and sequestered from the remaining constituents because of their potential destructive effects. This figure shows the two types of secretory pathways. The regulated secretory pathway, as its name implies, is a pathway for proteins that requires a stimulus or trigger to elicit secretion. Some stimuli regulate synthesis of the protein as well as its release. The constitutive pathway allows for secretion of proteins that are needed outside the cell, like in the extracellular matrix. It does not require stimuli, although growth factors may enhance the process.

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Finally, this cartoon also shows the packaging of lysosomes which will be discussed in more detail in a later section. For more information go to the lysosome web page.

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How does the Golgi complex regulate the insertion of plasma membrane proteins?

As you will recall from a previous presentation on rough endoplasmic reticulum we saw that they are inserted in the membrane at the level of the rough endoplasmic reticulum. The protein sequence is coded for membrane insert start and stop sites. This directed the insertion and alignment

points. Those that are multipass proteins have multiple start and stop sites.

The important role of the Golgi Complex is to make certain the plasma membrane proteins reach their destination. This figure shows the route. Note that the orientation of the protein is maintained so that the region destined to project outside the cell (a receptor binding site, for example), ends up in that place. In order to do this, it must be placed so that it faces inside the vesicle. Return to Menu

How does the Golgi complex add carbohydrate groups to a glycoprotein?

The Golgi complex is

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compartmentalized. Phosphorylation occurs in the Cis region. In other regions, different types of carbohydrates are added as a glycoprotein passes through the cisternae. This figure illustrates the different regions where sugars like mannose (man), galactose (gal), etc are added. The final sorting is done in the Trans Golgi complex.

The functional differentiation of the Golgi complex can be studied with the electron microscope with specific techniques that detect enzymes. The cis region is rich in lipid-bearing membranes and can be delineated by osmium tetroxide labeling. The middle regions label for enzymes that add carbohydrates or other groups on the product. The inner, or Trans region, is the area where the lysosomes are sorted. Therefore, it is heavily labeled for acid phosphatase.

There is much interest in understanding how the different Golgi cisternae are organized and differentiated. A number of models exist, however a favorite is called the "Maturational model" (Bannykh S.I. and Bakch, W.E. Membrane Dynamics at the Endoplasmic Reticulum Golgi Interface J Cell Biol 138: 1-4 1997)

This model suggests that the new vesicles from the ER enter the cis Golgi network and retrograde vesicles (bearing COPI) coats move to merge with the cis region cisternae.  These carry Golgi complex processing enzymes and their targeting to this region may be dependent on the low concentration of these processing enzymes.  Then, as processing continues, the middle cisternae contain more mature product and lower amounts of the enzymes needed in the beginning.  Finally, the trans region is specialized for sorting, containing receptors to sort and isolate lysosomal enzymes, for example.

For more information about modern studies that address this question, see: Wooding, S and H.R.B. Pelham, The dynamics of Golgi protein traffic visualized in living yeast cells. Molecular biology of the cell. 9: 2667-2680 1998

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Can proteins be transported back to the rough endoplasmic reticulum?

Sometimes vital proteins needed in the rough endoplasmic reticulum are transported along with the other proteins in the Golgi complex. The Golgi complex has a mechanism for trapping them and sending them back to the rough endoplasmic reticulum.

This cartoon shows the process. The protein destined for secretion is red. The blue protein must remain in the rough endoplasmic reticulum. The rough endoplasmic reticulum has inserted a receptor protein on the membrane it sends to the Golgi complex in the transitional vesicles (shown in green).  These are retrograde vesicles and are therefore coated with "COPI" (coatamer).  The ER protein receptor captures all of the  protein that carries the ER residency signal. . Vesicles then bud from the Golgi complex and move back to the rough endoplasmic reticulum. The receptor can circulate and continue to return the proteins needed by the endoplasmic reticulum.

A drug called "brefeldin A" blocks the transfer of protein to the Golgi complex, however the reverse transport is not blocked. The following electron micrograph shows the results of an experiment after a brefeldin A block.

For more information, see also, Cole, N.B., Ellenberg, J, Song, J, DiEuliis, D and Lippincott-Schwartz, J. Retrograde transport of Golgi-localized proteins to the ER. J Cell Biol 1-15, 1998.

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Receptor Mediated EndocytosisStructure-Function Correlations

Receptor mediated endocytosis is a process by which cells internalize molecules or viruses. As its name implies, it depends on the interaction of that molecule with a specific binding protein in the cell membrane called a receptor.

Read pages 618-633 and p 640 in your text: Alberts et al, Molecular Biology of the Cell, Garland publishing, N.Y., Third Edition, 1994.

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Internalization Events

Types of Ligands

Events in process

Patching and capping

Role of Clathrin Role of Adaptin Effect of temperature

Formation of Early Endosome

Formation of late endosome

Lysosomal development

Cholesterol entry as a model

Defects in trafficking

Types of receptors/pit

Regulated exocytosis

Protein packaging

 

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Test yourself!! How much do you know about receptor mediated endocytosis?

List the major types of internalization events?What types of ligands enter by receptor mediated endocytosis?Describe each step in the process ?What is the advantage of the patching and capping process to the cell?What is the role of the clathrin around the coated pit?What guides the ligand and receptor into the coated pit? What would happen if one

mutated the signal sequence on the receptor molecule?What is the effect of temperature on the process?  How do early endosomes form?  What is the Early Endocytic recycling pathway

and how is it used?What happens to the receptor in the endosome?  How do late endosomes form?How do late endosomes become lysosomes ?How does receptor mediated endocytosis reduce our serum levels of cholesterol?Do coated pits accommodate only one ligand, or can more then one enter in the

same pit?How are proteins destined for secretion sorted and packaged?What part of the Golgi complex produces secretory vesicles?Describe the process of Regulated exocytosis?What happens to the secretory vesicle membranes after exocytosis? Does the cell

simply get larger and larger because of the new membranes?In an electron micrograph, how would you distinguish profiles showing exocytosis

and endocytosis? Could both events be occurring at the same time?

How do cells internalize molecules and other cells?

This figure diagrams the major internalization events. In the two views on the right, receptors are not needed for internalization. During phagocytosis, cells may simply internalize particles or cells, like bacteria (cell eating). In the second, called pinocytosis, cells internalize soluble material (cell drinking). In both types of internalization, the cells extend processes and bring cells or soluble material into the cell in a vacuole. In the presentation on lysosomes , we learned that the vacuole

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formed in the cell by phagocytosis or pinocytosis often became a lysosome after hydrolases were brought to it and the pH was adjusted. The vacuoles formed are called phagosomes or macropinosomes. This cartoon was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

Endosomes are formed by receptor-mediated endocytosis. In this case, cells bring in proteins and other types of ligands attached to the plasma membrane via receptors. The process depends first on specific binding to the receptor, which is a subject worthy of a lecture in itself. This figure shows this process as "coated pit endocytosis". The coated pit is a specialized region of the membrane that is coated with clathrin (for stability, to aid the transport process). The coated pit forms a coated vesicle and then loses its clathrin coat. It then joins with other coated pits to form a receptosome.

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What types of ligands enter by receptor mediated endocytosis?

Toxins and lectins

Diptheria ToxinPseudomonas toxinCholera toxinRicinConcanavalin A

Viruses

Rous sarcoma virusSemliki forest virusVesicular stomatitis virusAdenovirus

Serum transport proteins and antibodies

TransferrinLow density lipoproteinTranscobalaminYolk proteinsIgE Polymeric IgAMaternal IgGIgG, via Fc receptors

Hormones and Growth Factors

InsulinEpidermal Growth Factor Growth HormoneThyroid stimulating hormoneNerve Growth FactorCalcitoninGlucagonProlactinLuteinizing HormoneThyroid hormonePlatelet Derived Growth FactorInterferonCatecholamines

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How does the process work?  An overview:

Receptors are brought to the plasma membrane by vesicles from the trans region of the Golgi complex . Review the definition of transmembrane proteins. Where and how are these receptor proteins inserted into the membrane? How does the Golgi complex maintain the fluidity of the plasma membrane , the receptors can move laterally in the membrane and collect in the specialized regions called clathrin coated pits.

Patching and capping

When the ligand binds to its specific receptor, the ligand-receptor complex accumulates in the coated pits. In many cells, these pits and complexes begin to concentrate in one area of a cell. Cytochemically, this appears as patches of label on the cell surface (patching) Eventually, the patches coalesce to form a cap at one pole of the cell (capping). Not all cells form caps, but most do form patches. Why would this process be an advantage for the cells? Imagine the large amounts of extracellular fluid that would be taken up if the cells endocytosed the ligand receptor complex all over its surface. Thus, the pre-concentration process minimizes the amount of fluid that is taken up in the vesicle.

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Effect of temperature on the process.

Recall the studies of membranes and Membrane fluidity . Receptors are moving in the plane of the membrane as long as the temperature is 37 C. In the presentation on Membrane fluidity, we talked about photobleaching with a laser beam. This allows you to study the lateral diffusion coefficients of the ligand-receptor complexes. Fluorescent molecules signal the site of the complexes and they are bleached after the laser exposure. Then, the photobleaching system measures the speed of the recovery in the bleached area (return of the fluorescence) as the label returns by lateral diffusion. This table was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985 An example of some measurements for different receptors is found in this table. The objective of the lateral movement is to collect the ligand-receptor complex in the clathrin coated pits. So, some receptors appear to be moving faster than others. One might speculate that this may be related to size of the receptor or the ligand. 

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Temperature may affect the binding of the ligand (rate) as well as the lateral mobility of the ligand-receptor complex. Some ligands will not bind well at low temperatures. However, others will bind, but not be taken in. This photograph shows the peroxidase (HRP) detection of a ligand that is distributed on the membrane at 4 C. Note the right hand control panel that shows absence of label in the presence of competing unlabeled ligand. Note the presence of the coated pit, even in the control. So, the formation of these is not temperature dependent. However, after warming for a few minutes, the formation of vesicles and endosomes is evident. It is important to note that Receptor mediated endocytosis is much faster than phagocytosis or pinocytosis. If one were to simply have a non-binding ligand present, it might take hours for the ligand to enter via pinocytosis. Thus, this rapid uptake coupled with the absence of label in the presence of competing ligand is a sign that this is receptor mediated. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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Some coated vesicles may be configured with a deep invagination, called a "neck". These were discovered by Willingham and Pastan and can be seen in serial sections as a thin region connection between the outside of the membrane and the vesicle. The vesicle contains labeled ligand attached to receptor. Formation of these necks is definitely temperature dependent as can be seen in the following table.

  

This figure and Table were taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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Finally, temperature is important to the overall patching and capping process discussed during the lecture on membranes. There we showed that, after they are bound, Membrane Receptors move laterally in the plane and groups of receptor-ligand complexes may actually coalesce in a patch and eventually in a cap. Antibodies are good examples of receptors that react this way. This figure shows what happens if the temperature is 4 C. There is a diffuse labeling. Warming the cells immediately produces patching. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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Formation and function of clathrin coated pits:

Clathrin coats surround the pit as diagrammed in the above cartoon. The assembly of the clathrin molecules on the pit appears to drive the pit to invaginate. This cage-like molecule may help stabilize the vesicle as it buds from the membrane. 

Clathrin coated pits may move in the plane of the membrane, however recent studies show

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that there is an "organized movement" as if the pits are tethered to cytoskeletal elements. The following paper studies coated pits in living cells that were transfected with a plasmid carrying a cDNA for green fluorescent protein (GFP) attached to the light chain of clathrin.  Gaidarov I, Santini, F, Warren, RA and Keen, JH Spatial control of coated-pit dynamics in living cells. Nature, Cell Biology 1: 1-7. 1999. The cells made GFP-clathrin and were able to insert the protein into coated pits.  This was tested via antibodies to coated pit proteins as well as studies of the endocytosis of transferrin. When time lapse photography was used to learn if the coated pits moved, they found that the pits appeared and disappeared at intervals.  Studies of regional spacing showed that appearance of new pits was often close to sites of old pits, suggesting regional organization. Superimposed images showed a linear pattern as if the pits were organized. 

The studies showed that the coated pits were resistent to detergents (Triton-X).   And, they were able to show that the retraction of cellular processes that followed triton-X treatment produced linear movement of each coated pit in the plasma membrane as if it was organized by or on cytoskeletal elements.  See the paper by this group (above citation) for the photographs and movies of these findings.

Formation of Endocytic vesicles.

Once the vesicle has formed, the clathrin coat is lost (perhaps via a chaperone protein of the heat shock protein 70 family). The loss of the coat is an energy requiring process. After the coat is lost, the vesicles join with other vesicles to form endosomes or receptosomes. The following electron micrograph shows clathrin coated pits forming a vesicle. It is taking up lipoprotein particles. Note how thick and well defined the clathrin coat is. This cartoon was taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. 1994, Third Edition

This micrograph was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985 Return to Menu

Role of adaptin in the transport of the receptor-ligand complex

How do receptors know how to get to the coated pits? Specific coa

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Continuation of studies of Receptor Mediated Endocytosis.

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Formation of the Early Endosome.

After entering the cytoplasm, the endocytotic vesicle loses its clathrin coat.  Then it quickly fuses with other such vesicles in a process called "homotypic" (same type) fusion.  Each vesicle has a pair of SNARE proteins, (a v-SNARE and a t-SNARE) which are used in the initial process (these are called cis-SNAREs).  In homotypic fusion, the act is primed by the untangling of the SNARE pairs by N-ethylmaleimide-sensitive factor (NSF) which also uses a Soluble NSF attachment protein (SNAP).

Then, the untangled t-SNAREs are stabilized by another factor called LMA-1. The t-SNARE of one vesicle is aligned with the v-SNARE of another. In the meantime, specific docking/tethering factors bring the vesicles into closer alignment using their surface rab5-GDP. One of these is Rabaptin-5, which occurs with a nucleotide exchange factor (Rabex-5) that can activate rab5. It also has two  GTPase that could tether two endosomes together.   Another factor is Early Endosome Associated Protein (EEA-1) which are "coiled coils'.  Its C terminus has a FYVE finger domain that binds phosphatidylinositol-3-phosphate in endosome membranes.  EEA-1 not only tethers endosomes, it activates endosome fusion. This process is seen in the following cartoon

Thus, markers for early endosomes include:

EEA-1 proteins rab5-GDP pH around 6.0

.

 

 

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Early endosome recycles receptors to the surface

Early endosomes have lowered pH (5.9-6) and this can release the receptor and ligand.  The receptor may be recycled to the surface by vesicles that bud from the endosome and then target the plasma membrane. After these recycling vesicles fuse with the plasma membrane, the receptor is returned to the cell surface for further binding and activity.  Then, the early endosome converts to a late endosome.

What happen to each receptor in the endosome?

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The exact fate of the receptor in the membrane appears to vary with the cell. It can also be degraded. However, some receptors move to the Golgi complex to be added back to membranes in the Trans Golgi region. This would recycle the receptor. This process is similar to the process by which lysosomal enzyme receptors are recycled. In many cases, the receptor is sent back to the plasma membrane after a transport vesicle buds from the endosome. This event is shown in the above photograph. Willingham and Pastan used ferritin labeled antibodies to the extracellular domain of the receptor to follow recycling (transferrin receptor). This photograph was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

As stated in the above paragraphs, in cases where the receptor is recycled from a budding endosome, the endosome itself is called an "early endosome". In other words, the pH has dropped just enough to allow the ligand to drop off, however, the receptor is not degraded. This keeps it intact so it can be recycled back to the membrane.

 

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Formation of a late endosome

Late endosomes are formed as the pH continues to drop to 5-6.0.  Also, clathrin-coated vesicles from the Trans Golgi Network carry digestive enzymes to the late endosome and fuse with these structures, releasing their contents. The late endosome thus becomes a degradative body and also acquires the marker for mannose 6 phosphate receptor "MPR+

It changes its rab surface marker to rab7-GTP, probably to accommodate the new targeting vesicles with which it will fuse. This means that the late endosome can be identified by the presence of the rab7.

Late endosomes include multivesicular bodies and contain whorls or vesicles of membranes inside. They also contain an unusual lipid which has become another marker for this stage.  The lipid is called lysobisphosphatidic acid (LBPA) which has a larger head group than tail. Its structure enables it to be inserted into highly curved membranes, like the membrane whorls.  It is believed that this allows retention and binding of specific molecules in the whorls by lipid-protein; lipid-lipid interactions.  One type of molecule is cholesterol and it is believed that this is an important site for cholesterol accumulation.

For more information about the role of LBPA, see: Kobayashi, T, Beuchat, M.H, Lindsay, M, Frias S, Palmiter, R.D., Sakuraba, H, Parton, R.G, and Gruenberg, J.  Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biology 1: 113-118. 

Late endosomes function to degrade many proteins and lipids.  They also are responsible for returning the MPR receptors back to the Trans Golgi network.  They recycle these by budding off membranes that carry back the receptors and target the Trans Golgi membranes for fusion.  After fusion, the MRP receptors are available to capture and sort new degradative enzymes for future trafficking to the late endosomes.

To summarize, markers for late endosomes include:

rab7-GDP LBPA MPR+

Late endosomes fuse with lysosomes.

Finally, late endosomes may not be able to digest all the material. Therefore, the next step is a fusion of late endosomes and lysosomes creating a hybrid organelle.  Residual heavily glycosylated lysosomal associated membrane proteins (LAMPs) may thus be transmitted to lysosomes.  LAMPs then become a marker for a late endosome or a lysosome. Since lysosomes do not have MPR receptors (they have all been sent to the Golgi), one could distinguish the lysosome and the late endosome on the basis of labeling for MPR.  Thus, fusion begins after the MPR have been sent back to the Trans Golgi.

The steps involved in forming late endosomes and lysosomes are drawn in this cartoon. Note that lysosomes continue to communicate with late endosomes and may deliver important material back to this group of organelles.  Lysosomes are considered the end product of endocytosis.  Thus,  lysosomes do not communicate directly with the Trans Golgi (and hence

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the plasma membrane).  However, they could communicate with upstream structures by way of the retrograde transport to the late endosome.

To summarize, markers for lysosomes include:

LAMP+ acid hydrolases MPR negative NPC1 (in normal cells)

For more information, see: Mukherjee , S, and Maxfield, F.R. Cholesterol: stuck in traffic.  Nature Cell Biology 1: E37-E38.

Receptor-mediated uptake of LDL receptors: a model for studies of trafficking and defects.

Cholesterol bound to Low density lipoproteins (LDL) is taken up by cells so that cholesterol can be used in construction of membranes, etc.  In this case the receptor is recycled and the ligand (LDL-cholesterol) is metabolized so the free cholesterol can be released and used by the cell. There are two genetic mutations that cause either no uptake of LDL receptors or uptake and accumulation of cholesterol in late endosomes.  We will look at these diseases to learn more the importance  of Key elements in receptor mediated endocytosis are.

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First, this cartoon summarizes the entire process of endocytosis of LDL-cholesterol bound to LDL receptors. (taken from Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994 ). To review, after the ligand and receptors are collected in the coated pits, which then form coated vesicles, the clathrin coat is removed and the vesicle fuses with forming endosomes. The early endosome allows recycling of the LDL receptor.  The late endosome/lysosome is the site of accumulation of cholesterol followed by hydrolysis.  Free cholesterol is then available to be used by the cell.   

How do receptor mediated endocytosis and LDL receptors help reduce our serum levels of cholesterol?

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The above cartoon shows a figure from your text in which the LDL receptors are collecting with their ligand (LDL) in a clathrin coated pit. LDL has been called the "bad cholesterol". High serum LDL's go along with high serum cholesterol However, we can reduce serum cholesterol by taking it up into cells that need it (for membranes, steroid hormone production, etc.) This requires a specific LDL receptor and a working receptor mediated endocytosis process. 

Some families have a defect in the Adaptin-2 binding site on the LDL receptor.  Recall that this site helped concentrate the LDL receptor in the coated pit.  In fact, binding to the Adaptor protein actually helped recruit the clathrin to the site.  The defect is shown in the above cartoon from your text. This genetic deficiency  prevents LDL and its receptor from entering the coated pit or from being taken up. The result very high serum levels of cholesterol and all the problems resulting from that. 

This cartoon was taken from Alberts et al. Molecular Biology of the Cell, Garland Publishing, Third edition, 1994.

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Cholesterol Stuck in Traffic: What we can learn from studies of Nieman Pick type C disorder.

Recent studies point to an important protein involved in cholesterol efflux from the late endosomes, called NPC1.  This protein has a putative steroid binding domain. It may be involved in transport of cholesterol to the Trans Golgi Network and then to plasma membranes.  Hence it may be important in the addition of cholesterol to membranes where it is needed.  It may also be a sensor for sites of accumulated cholesterol.

It got its name from a disease, which is caused by an autosomal recessive mutated gene in which this NPC1 protein is not normal.  This is called Niemann Pick type C disorder. The mutation results in an accumulation of cholesterol in late endosomes which also expand, filling with whorls of membranes.   

Where is NPC1 protein normally found?  Studies by Neufeld et al (J Biol Chem. 274: 9627-9635 (1999)  have shown however that NPC1 protein in normal fibroblasts is in the lysosomes (identified by their LAMP marker and absence of MPR).  Yet, this same compartment did not contain large amounts of LDL-cholesterol.  Thus, Neufeld et al  suggested that lysosomes send the NPC1 back to late endosomes by retrograde traffic (see above cartoon).  Perhaps this is stimulated by the concentration of cholesterol in the late endosomes.  As Mukherjee and Maxfield ( Cholesterol, Stuck in Traffic, Nature Cell Biology 1: E37-E38) point out, this route would then give direct access to the Trans Golgi network to allow trafficking of cholesterol to sites where the cholesterol is needed.  Lysosomes don't have routes to these sites, so they may have to communicate to the rest of the cell via late endosomes.

Where is cholesterol needed?  Wherever there is a need for more structural stabilization of membranes, for example wherever there are coated pits, particularly clathrin coated pits.

Kobayashi et al [Kobayashi, T, Beuchat M-H, Lindsay, M, Frias S, Richard D. Palmiter, H

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Sakuraba, R.G. Parton, and J. Gruenberg  Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biology 1: 113-116 (1999).] studied fibroblasts from Nieman Pick Type C disorder (NPC) and found that not only was cholesterol stuck in the late endosome, mannose 6 phosphate receptors (MPR) are also stuck in this part of the trafficking pathway.  Immunolabeling for MPR and CD63 (a marker for late endosomes) showed normal distribution in late endosomes and Golgi regions in normal cells as well as in cells from another disease state, Tay Sach's.  However,  cells from patients with NPC had cholesterol and MPR almost exclusively in late endosomes.  

These studies continued by using a drug (U18666A) to mimic the disease state and they found that they could cause both an accumulation of the cholesterol in late endosomes, and also a block in the transport of MPR back to the Golgi complex.  Hence, MPR was detected exclusively with structures labeled for late endosome markers including LBPA and rab7.

What is the significance of these studies?  The NPC1 protein may function as a cholesterol sensor/binding protein. An accumulation of cholesterol in the late endosomal compartment may stimulate retrograde transport from lysosomes to endosomes bringing the NPC1 protein to the endosomes.  There, the cholesterol can be sorted and further distributed to membranes throughout the cell, as needed.  If the NPC1 protein is not functional, then cholesterol accumulates in the membrane whorls seen the late endosomes.  This appears to block retrograde transport from the endosome to the Golgi complex, as evidence by the accumulation of the MPR.  Hence, not only is cholesterol "stuck in traffic", so is the mannose 6 phosphate receptor (MPR).  It appears to be stuck as a consequence of the cholesterol accumulation.   There is too little known about the return trip to the Golgi complex. However, stay tuned for future information about the role of microtubules and associated motor proteins.  

In the meantime,  Mukherjee and Maxfield (Nature, Cell Biology, 1: E37-E38 (1999)  speculate that perhaps the accumulation of cholesterol in the late endosome makes the membranes less elastic and thus, the return vesicles to the Trans Golgi Network cannot bud and form.  There may be broader effects on trafficking as well.  Return to the page on membrane architecture for more information about the role of cholesterol in membrane

structure and function.

Below is a modified cartoon showing the site of the block in trafficking in Nieman Pick C disorder.

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Do coated pits accommodate only one ligand, or can more than one enter at a time?

Supposing a cell is stimulated with two hormones (and it has receptors for both), or a hormone and a growth factor. Do both ligands enter via the same packages, or is there a selection for one particular type of ligand in a coated pit. This is a perfect question for cytochemistry with different types of labels attached to different ligands. For example, one

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can use colloidal gold, ferritin, peroxidase and detect as many as 2-4 ligands on a given cell. 

This figure shows that multiple ligands can enter the cell in the same coated pit. Furthermore, the vesicles will carry them to the same receptosomes. The photo shows co-detection of ligands as diverse as Epidermal growth factor, vesicular stomatitis virus, or alpha 2 macroglobulin. Labeling molecules (signalling molecules) included gold, peroxidase, ferritin, or the virus itself. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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How are proteins destined for secretion sorted and packaged.

This cartoon shows the sorting of proteins destined for secretion into vacuoles in the trans Golgi Complex. Note that lysosomal enzymes are sorted into another compartment, thanks to the mannose 6 phosphate

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receptor. For review, see the 

What is Regulated exocytosis ?

The above cartoon also shows the process of exocytosis. We defined regulated secretion initially when we talked about the Golgi complex . Regulated secretion requires a stimulus, usually from the outside. The process may begin by the binding of a ligand to a specific receptor. This activates the receptor which turn activates a cascade of events (second and third messengers) leading to the release of the product. If the product is stored in a secretory granule, it is released by a process called "exocytosis". Therefore, the process of receptor mediated endocytosis and exocytosis can and does occur simultaneously. One should be able to recognize the cytological signs of both processes in the same cell. Click this link to see an electron micrograph of exocytosis.

Distinguish profiles showing exocytosis and endocytosis

As we said earlier in this presentation, clathrin coated pits serve like a flat "basket", stabilizing the area to be

internalized. They are not exclusive to the plasma membrane. However, it is important to be able to distinguish them from exocytosis profiles associated with the plasma membrane.For example, this electron micrograph is showing the process of exocytosis . The process begins by fusion of the membranes at the peripheral pole of the granule. Then an opening is created which widens to look like an omicron figure. This opening allows the granular material to be released. The membrane is now part of the plasma membrane and any proteins carried with it can be incorporated into the plasma membrane. Note that there is no coating on the membrane. This figure was taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing Third Edition, 1994

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In contrast, this micrograph shows a figure which looks something like an omicron, however, this view is showing receptor mediated endocytosis of virus particles. In both cases, the membrane is coated with clathrin and these represent classical receptor mediated endocytosis profiles. Most ligands cannot be visualized by themselves, like a virus particle. Therefore, the cytochemist must attach label to the ligand. Alternatively, the cytochemist could immunocytochemically detect the receptor with antibodies that recognize the extracellular domain. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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How are lysosomes and peroxisomes produced?

What are lysosomes?

Lysosomes are the cells' garbage disposal system. They degrade the products of ingestion, such as the bacterium that has been taken in by phagocytosis seen in the above cartoon. After the bacterium is enclosed in a vacuole, vesicles containing lysosomal enzymes (sometimes called primary lysosomes) fuse with it. The pH becomes more acidic and this activates the enzymes. The vacuole thus becomes a secondary lysosome and degrades the bacterium.

Lysosomes also degrade worn out organelles such as mitochondria. In this cartoon, a section of rough endoplasmic reticulum wraps itself around a mitochondrion and forms a vacuole. Then, vesicles carrying lysosomal enzymes fuse with the vesicle and the vacuole becomes an active secondary lysosome.

A third function for lysosomes is to handle the products of receptor-mediated endocytosis such as the receptor, ligand and associated membrane. In this case, the early coalescence of vesicles bringing in the receptor and ligand produces an endosome. Then, the introduction of lysosomal enzymes and the lower pH causes release, and degradation of the contents. This can be used for recycling of the receptor and other membrane components. See the Web page on Receptor mediated endocytosis for more information.

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Lysosomes carry hydrolases that degade nucleotides, proteins, lipids, phospholipids, and also remove carbohydrate, sulfate, or phosphate groups from molecules. The hydrolases are active at an acid pH which is fortunate because if they leak out of the lysosome, they are not likely to do damage (at pH 7.2) unless the cell has become acidic. A Hydrogen ion ATPase is found in the membrane of lysosomes to acidify the environment.

Lysosomal morphology varies with the state of the cell and its degree of degradative activity. Lysosomes have pieces of membranes, vacuoles, granules and parts of mitochondia inside. Phagolysosomes may have parts of bacteria or the cell it has injested. This electron micrograph shows typical secondary lysosomes. They have been detected by cytochemical labeling for acid phosphatase. This is a good marker for lysosomes. Recall that it is also used as a marker for the Trans Golgi Cisternae.

How does the Golgi Complex sort lysosomal enzymes?

The Golgi complex sorts the lysosomal enzyme in the Trans region. It is received from the rough endoplasmic reticulum (RER in this cartoon) in the cis region.

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There it has a phosphate radical attached to the mannose residue. This mannose-6 phosphate forms a sorting signal that moves through the cisternae to the trans region where it binds to a specific receptor. After it binds to the receptor, it begins to bud and a "cage" or "coat" made of clathrin forms around the bud (to strengthen it). It moves away to fuse with a developing lysosome (such as the vacuoles seen in the previous figure). This lysosome contains a hydrogen ion pump on its surface. The pump works to acidify the environment inside the lysosome. This removes the phosphate and dissociates the hydrolase from the receptor. The receptor is then recycled back to the Golgi complex

Lysosomes can actually be detected by pH indicator dyes. This photograph shows dyes that indicate different pH's with different colors. The red lysosomes (pH 5.0) are probably typical lysosomes. The blue and green lysosomes are probably endosomes. This change can be detected if you link a ligand to fluorescein. Fluorescein will not fluoresce at pH's lower than 6.0. Therefore, one can follow entry of the receptor-ligand complex and then see the fluorescence disappear as the endosome containing the complex is acidified.

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Why peroxisomes are not like lysosomes.

Peroxisomes are organelles that contain oxidative enzymes, such as D-amino acid oxidase, ureate oxidase, and catalase. They may resemble a lysosome, however, they are not formed in the Golgi complex. Peroxisomes are distinguished by a crystalline structure inside a sac which also contains amorphous gray material. They are self replicating, like the mitochondria. Components accumulate at a given site and they can be assembled into a peroxisome. They may look like storage granules, however, they are not formed in the same way as storage granules.  They also enlarge and bud to produce new peroxisomes.

Peroxisomes function to rid the body of toxic substances like hydrogen peroxide, or other metabolites. They are a major site of oxygen utilization and are numerous in the liver where toxic byproducts are going to accumulate.

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