8/12/2019 Outline Ch 08

1/21

CHAPTER8

CYTOPLASMIC MEMBRANE S YSTEMS : S TRUCTURE ,

FUNCTION , AND MEMBRANE TRAFFICKING

An Overview of the Endomembrane System and Its Dynamic Nature

I. Before the 20th century - stained tissue sections hinted at an extensive membrane network in cytoplasmA. 1940s - EM revealed diverse array of membranous structures in cytoplasm of most eukaryotes

1. Membrane-bound vesicles of varying diameter; containing material of different electron density2. Long channels bounded by membranes that radiate through cytoplasm; form an interconnected

network of canals3. Stacks of flattened, membrane-bound sacs (cisternae)

B. These studies & subsequent biochemical studies showed that eukaryotic cell cytoplasm was subdividedinto a variety of distinct membrane-bound compartments1. Saw distinct organelles in diverse cells from yeast to higher plants and animals2. The organelles may appear as stable structures, but, in fact, they are dynamic compartments that are

in continual flux3. These organelles have distinct structures & functions but together form an endomembrane system;

the individual components function as part of coordinated unitC. Mitochondria & chloroplasts are not part of this interconnected systemD. Current evidence suggests that peroxisomes have a dual origin

1. The basic elements of the boundary membrane are thought to arise from the endoplasmic reticulum,..

2. But many of the membrane proteins & soluble internal proteins are taken up from the cytoplasm

II. These organelles are part of dynamic, integrated network; materials are shuttled between parts of cellA. Transport vesicles shuttle things between organelles; form by budding from donor compartment

1. Vesicle implies a spherical-shaped carrier; cargo may also be transported in irregular or tubularshaped membrane-bound carriers2. But the term vesicle is often used, keeping in mind that they are not always spherical

B. Transport vesicles move in directed manner, often pulled by motor proteins operating on tracks formedby microtubules & microfilaments of the cytoskeleton

C. When they reach their destination, they fuse with acceptor compartment, which receives vesicles' solublecargo & membrane wrapper

D. Exhibit repeated cycles of budding & fusion that move a diverse array of materials along numerouspathways traversing the cell

III. Several distinct pathways through cytoplasm have been identified; they fall into two groups: a biosynthetic(secretory) pathway & an endocytic pathway

IV. Biosynthetic (secretory) pathway synthesis in ER (protein) or Golgi (lipid, carbohydrate); altered as passthroughGolgi, sent fromthere to various locations(membrane, lysosome, largeplantcell vacuole, etc.A. Many materials made in ER (proteins)&Golgi (complexpolysaccharides) fated for secretion from cellB. Two types of secretory activity - constitutive & regulated

1. Constitutive - synthesis & secretion into extracellular space occurs in continual, unregulated manner;most cells do it to form extracellular matrix & plasma membrane itself

2. Regulated - secretory materials are often stored in large, densely packed, membrane-bound secretorygranulesin cell periphery; secreted after correct stimulus

65

8/12/2019 Outline Ch 08

2/21

a. Endocrine cells release hormonesb. Pancreatic acinar cells release digestive enzymesc. Nerve cells release neurotransmitters

C. Proteins, lipids & complex polysaccharides are transported through cell along biosynthetic or secretorypathway; discussion will center on several distinct classes of proteins1. Soluble proteins discharged from cell

2. Integral proteins of various membranes3. Soluble proteins that reside within various compartments enclosed by endomembranes (like lysosomalenzymes)

V. Endocytic pathway - moves materials or membrane surface into cell from outside to cytoplasmiccompartments (endosomes, lysosomes); movement direction is opposite to that of secretory pathway

VI. Proteins targeted to specific destinations through sorting signals located on them & receptors in transportvesicle walls that recognize them (analogous to trucks carrying different cargo to various sites)A. Both transport pathways require defined traffic patterns; ensure accurate delivery to correct sites

1. Ex. - salivaryglandcell proteintrafficking;salivary mucus proteins (made in ER) specifically targeted tosecretory granules; lysosome enzymes (also made in ER) specifically sent to lysosome

2. Different organelles also contain different integral membrane proteins; they must also be targeted toparticular organelle (lysosome, Golgi cisterna)

B. Targeting involves integral membrane proteins, secretory proteins, lysosomal proteins; they are routed totheir appropriate cellular destination by virtue of specific addresses (sorting signals)1. Sorting signals are encoded in protein amino acid sequence or in the attached oligosaccharides

C. Sortingisfacilitatedbyspecific membrane or surface coat receptors for sorting signals found in particularendomembranemembranes of endomembrane system or by coats that form on outer surfaces of transportvesicles1. Specific receptors reside on surface coats or in the membranes of budding vesicles2. Ensures that protein is transported to the appropriate destination3. For most part, machinery responsible for driving this complex distribution system consists of soluble

proteins that are recruited to specific membrane surfacesD. Great advances in experimental approaches have been made over last 2 or 3 decades in:

1. Mapping the traffic patterns that exist in eukaryotic cells2. Identifying the specific addresses & receptors that govern the flow of traffic3. Dissecting the machinery that ensures that materials are delivered to appropriate cellular sites

A Few Approaches to the Study of Cytomembranes

I. EM micrographs give detailed view of cell cytoplasm, but little insight into functions of the structuresA. Cells perform dynamic processes, but EM portrays only static scenesB. Determining functions of cell organelles required new techniques & innovative experiments

II. Insights gained from autoradiography - can detect location of radioactively labeled materials in cellA. Pancreas acinar cells have a particularly extensive endomembrane system; ideal for study by

autoradiography1. The cells function primarily in synthesis & secretion of digestive enzymes2. Enzymes are shipped via ducts from pancreas, where they are synthesized, to small intestine to

degrade ingested food matterB. James Jamieson & George Palade (Rockefeller U.) - worked with pancreas acinar cells

1. Followed secretory protein from synthesis to secretion & determined individual steps even though all ofthem occurred simultaneously

2. Able to observe steps of single cycle of secretion from start to finish

66

8/12/2019 Outline Ch 08

3/21

3. Autoradiography allows visualization of biochemical processes by allowing investigator to determinethe location of radioactively labeled materials within cell

C. Procedure - section tissues containing radiolabel & locate hot digestive enzymes with autoradiography1. Incubatetissuesliceswith hot (radioactive) aminoacids briefly>incorporatedintodigestiveenzymes

astheyaremade on ribosomes2. Fix tissues; tissue sections containingradioactive isotopes were then covered with thin photographic

emulsion layer, which is thus exposed to radiolabel emanating from radioisotopes within tissue3. Sites in cell with radiolabel are highlighted with developed silver grains in overlying emulsion4. If label, wash & harvest immediately, label appears first over RER > RER was site of synthesis

III. Insights from pulse-chase trials (Palade & Jamieson) - show secretory protein path after synthesis to their siteof dischargeA. Expose to hot amino acids briefly (pulse) followed by a wash to remove excess isotope from tissue

1. Pulse refers to the brief incubation with radioactivity during which labeled amino acids are incorporatedinto protein

B. Transferred tissue to medium with unlabeled amino acids (chase), which lasts for varying time periods1. During this period, protein synthesis continues using nonradioactive amino acids2. The longer the chase, the farther the hot (radioactive) proteins made during the pulse will have traveled

from their synthesis site (the RER) within the cellC. One can see wave of radioactivity moving through cell, discern pathway sequence - RER was synthesis site

& see rest of pathway from one location to the next until the process is complete1. Defined the secretory (biosynthetic) pathway & tied a number of seemingly separate membranous

compartments into an integrated functional unit

IV. Insights gained from use of green fluorescent protein (GFP) scientists can follow within their own eyes thedynamicmovements of specificproteins as they move within single living cell; do not have to kill cellsA. GFP is small protein from certain jellyfish that emits a green fluorescent light

1. Its gene has been isolated & can be fused to DNA encoding protein to be studied2. The resulting chimeric (composite) DNA is introduced into cells that can be observed in scope3. Once inside cell, chimeric DNA expresses chimeric protein consisting of GFP fused to end of protein to

be studied

4. Usually, GFP stuck to end of a protein has little or no effect on its movement or function & proteinunder study has no effect on fluorescence of attached GFP

B. Example: infect cells with vesicular stomatitis virus (VSV) strain in which a viral gene (VSVG) is fused toGFP gene; viruses useful since they turn cells into factory for producing viral proteins1. These viral proteins are carried like any other protein cargo through the biosynthetic pathway2. Cell begins to make massive amounts of VSVG protein in RER3. VSVG then goes to Golgi complex & eventually to the plasma membrane of the infected cell where the

are incorporated into viral envelopes4. Cansee relatively synchronouswaveof proteinmovement(greenfluorescence)soonafterinfection5. Synchrony is enhanced by use of virus with mutant VSVG protein that cannot leave ER of infected cell

grown at elevated temperature (40C)6. When temperature is lowered to 32C, the fluorescent GFP-VSVG protein that had accumulated in ER

moves synchronously to Golgi complex for various processing events & then to membrane7. Mutants of this type that function normally at reduced (permissive) temperature, but not at elevated(restrictive) temperatures are described as temperature-sensitive mutants

V. Insights gained from the biochemical analysis of subcellular fractions - cell homogenization & organelleisolation techniques were pioneered by Albert Claude & Christian De Duve (1950s & 1960s)A. Homogenize cells; form cytoplasmic membrane fragments, the ends of which fuse to form spherical

vesicles (

8/12/2019 Outline Ch 08

4/21

B. Vesicles formed from different organelles (nucleus, mitochondrion, plasma membrane, ER, etc.) havevaried properties, which allow their separation (cell fractionation) from one another1. Endomembrane system (primarily ER, Golgi) vesicles form heterogeneous, similar-sized vesicles

(microsomes); rapidly (& crudely) purified, then separated further; often retain biological activity2. Fractionatemicrosomes into smooth & rough membrane fractions by gradient techniques (Ch. 18)3. Once isolated, one can determine the biochemical composition of various fractions

C. Example of uses & findings - vesicles from different parts of Golgi were found to have enzymes that adddifferent sugars to the ends of growing CHO chains of glycoprotein or glycolipids1. Purify these enzymes from the microsomal fraction; use them as antigens to make antibodies & attach

gold particles to the antibodies, locations of which in Golgi membranes can be seen in EM2. Revealed role of Golgi complex in stepwise assembly of complex carbohydrates

D. Example: identification of proteins in cell fractions taken to new level using sophisticated proteomictechnology; isolate organelle, extract & separate proteins & then identify them by mass spectrometry1. Hundreds of proteins can be identified simultaneously, providing a comprehensive molecular portrait o

any organelle that can be prepared in a relatively pure state2. For example, a simple phagosome, containing an ingested latex bead had >160 different proteins, many

of which had never before been identified or were not known to be involved in phagocytosis3. Several proteins were included that were characteristic of ER, leading to new appreciation of the ER's

role in phagocytosis

VI. Insights gained from use of cell-free systems isolated parts of cell studied for their capabilitiesA. These cell-free systems (which do not contain whole cells) provide information about complex processes

that were impossible to study using intact cellsB. George Palade, Philip Siekevitz, et al. (Rockefeller University, 1960s) studied properties of rough

microsomal fraction1. Stripped rough microsomal preparation of its attached particles & found that isolated particles

(ribosomes) could synthesize proteins when provided with the required cytosol ingredients2. Newly synthesized proteins were released into the aqueous fluid in test tube3. When same experiments were conducted with complete rough microsomal fraction, the proteins were

not released into incubation medium but were trapped within membranous vesicle lumens4. So microsomal membrane was not needed for protein synthesis, but for sequestering newly made

secretory proteins within ER cisternal spaceC. Over the past few decades, cell-free systems have been used to identify the roles of many of the proteins

involved in membrane trafficking; example below of budding from liposomes1. Cell-free liposomes (vesicles whose walls consist of an artificial bilayer created from purified

phospholipids) used to study specific roles of proteins involved in budding2. Incubate liposomes with purified proteins that normally comprise coats of cell transport vesicles3. Without added coat proteins > no vesicle budding; with it > get budding4. Such reconstitution of cellular processes in vitrofrom purified components has been useful in this &

other studies like..a. Determining the proteins that bind to the membrane to initiate vesicle formationb. Determining those proteins responsible for cargo selection &c. Determining those proteins that sever the vesicle from the donor membrane

VIII. Insights gained from study of mutant phenotypes a mutant is an organism (or cultured cell) whosechromosomes contain one or more genes that encode abnormal proteinsA. Mutant gene products vary from the normal; they can cause a characteristic deficiency in the cell carrying

the mutation, which is analyzed1. Determining the precise nature of deficiency gives information on function of the normal protein

B. Randy Schekman, et al., Univ. of Ca. Berkeley studied genetic basis of secretion using yeast cells

68

8/12/2019 Outline Ch 08

5/21

1. Why he used yeast cells - few genes, small, single-celled & able to be cultured easily, can be grown ashaploid so mutants seen; haploid for majority of life cycle; allows easier deficiency detection

2. Gene mutation in haploid yields observable effect; cant mask presence of abnormal gene withnormal one

3. Yeast ER simple & directly connected to outer membrane of nuclear envelope; vesicles bud from ER,travel to Golgi cisternae where they fuse

4. Find genes involved in secretory pathway by screening for mutant cells with abnormal distribution ofcytoplasmic membranes (SECgenes)5. Found mutation in gene for protein involved in vesicle formation at ER membrane > in absence of

vesicle formation, cells accumulated expanded ER cisternae6. Found another mutation in gene encoding a protein involved in vesicle fusion > if this gene is

defective, cells amass an excess number of unfused vesicles7. Many mutants that disrupt secretory pathway have been found, cloned & sequenced; mutant proteins

have been isolated; homologous proteins (with related sequences) found in mammals

IX. Lessons learned from these techniquesA. Dynamic activities of endomembrane systems are highly conservedB. Processes similar in all organisms (yeast, plant, insect & human cells); done with remarkably similar

proteins (despite their structural diversity, these cells have underlying molecular similarities)1. Someproteins doingsimilar things in different (often widely divergent) species are interchangeable2. Mammalian cell-free systems can often use yeast proteins to facilitate vesicle transport3. Researchers can "cure" yeast biosynthetic pathway mutants by genetically engineering them to carry

normal mammalian genes

The Endoplasmic eticulum !E"# $ac%&round Information and 'eneral

Functions

I. History and general description - first detected in 19th centuryA. Vague cytoplasmic network seen in stained cells (ergastoplasm)

1. In pancreas cells, ergastoplasm seen to disappear upon starvation & reappear when animal fed2. Concluded ergastoplasm in pancreas makes digestive juices

B. Later seen in EM by Porter who renamed it endoplasmic reticulum

II. Endoplasmic reticulum (ER) is divided into 2 broad categories - rough & smooth; both enclose space socytoplasm divided into cytosolic&luminal (or cisternal) space; contents of the 2 spaces arequite differentA. Fluorescently labeled proteins & lipids can diffuse from one type of ER into the other, indicating that their

membranes are continuous1. The 2 types of ER share many of the same proteins & engage in certain common activities (synthesis of

certain lipids & cholesterol)2. At the same time, however, numerous proteins are found only in one or the other type of ER3. Thus, RER & SER have important structural & functional differences, which can be traced to the

presence of different proteins in the 2 compartmentsB. Smooth ER (SER) - typically tubular; interconnecting pipeline system; curves through cytoplasm; lacks

associated ribosomes1. Membranous elements of the SER are highly curved & tubular, forming an interconnecting system ofpipelines curving through the cytoplasm

2. When cells are homogenized, it fragments into smooth-surfaced vesiclesC. Rough ER (RER) extensive organelle defined by presence of ribosomes boundto its cytosolic surface; ma

mostly of cisternae (interconnected network of flattened sacs); space inside appears continuous1. RER is continuous with nuclear envelope outer membrane (it has ribosomes on cytosolic surface)2. When cell is homogenized, RER fragments into rough-surfaced vesicles

69

8/12/2019 Outline Ch 08

6/21

3. Because they have different densities, rough & smooth vesicles can be readily separated by densitygradient centrifugation & then studied

D. Different cell types contain varying amounts of either one ER type or other; depends on cell activities1. Cells that secrete large amounts of proteins (pancreas or salivary gland cells) > lots of RER

III. Smooth ER functions - extensively developed in many cells (skeletal muscle, kidney tubules, steroid-

producing endocrine cells); its specific proteins vary cell-to-cell depending on functions of cells SERA. Synthesis of steroid hormones in gonad & adrenal cortex endocrine cellsB. Detoxification in liver of many organic compounds (barbiturates & ethanol), whose chronic use can lead to

SER proliferation in liver cells; detoxification carried out by oxygen-transferring enzymes1. These oxygenases, like cytochrome P450s, convert these compounds into more hydrophilic

derivatives so that they can be more easily & readily excreted2. Sometimes the oxygenases create carcinogens; relatively harmless benzo[a]pyrene formed when meat

charred on a grill is converted into potent carcinogen by SER detoxifying enzymes3. Such enzymes have low substrate specificity; oxidize 1000s of different hydrophobic compounds4. Cytochrome P450smetabolize many prescribed medications; genetic variation in these enzymes among

humans may explain differences between people in drug effectiveness & side-effectsC. Sequestering Ca2+ions within the cytoplasm of cells inside the cisternal space; release of these Ca2+ions

triggers specific cell activities1. SER contains a high concentration of Ca2+-binding proteins2. Regulated Ca2+ion release from SER of skeletal & cardiac muscle cells triggers specific cell responses,

like skeletal muscle cell contraction & fusion of secretory vesicles with plasma membrane3. SER in skeletal & cardiac muscle cells is known as the sarcoplasmic reticulum

III. Rough ER functions - predominantly export or membrane protein synthesis (pancreatic acinar cells, mucus-secreting cells of digestive tract lining; early studies done on these cells)A. Organelles of protein-secreting, glandular epithelium cells are distinctly polarized along cell tall axis (from

basal to apical end); reflects flow of secretory products from synthesis to discharge1. Nucleus & extensive RER cisternae found near cell basal surface near blood supply; RER is site of

synthesis proteins, carbohydrate chains & phospholipids that move through cytomembrane system2. Golgi complex is located in central region of cell

3. Apical surface faces duct lumen that will carry secretory product out of organ4. Cell apical end contains membrane-bound secretory vesicles whose contents are released upon arrivalof appropriate signal

B. It was found that RER is secretory protein synthesis site (starting point of biosynthetic pathway) inpancreatic acinar cells1. Other examples found later - intestinal goblet cells (secrete mucoproteins), endocrine cells

(polypeptide hormones), plasma cells (antibodies), liver cells (blood serum proteins)

The Endoplasmic eticulum !E"# Synthesis of (roteins on )embrane*$ound vs+

Free ibosomes

I. Further experiments revealed that polypeptides are synthesized at 2 distinct locales within cell

A. Some proteins are made on ribosomes attached to cytosolic surface of RER membranes1. Proteins secreted from cells2. Integral membrane proteins3. Soluble proteins that reside within compartments of endomembrane system (ER, Golgi complex,

lysosomes, endosomes, vesicles, plant vacuoles)B. It is estimated that roughly one-third of all polypeptides encoded by the human genome are synthesized on

free ribosomes (not attached to ER) & then released into cytosol, including:1. Proteins destined to remain in cytosol (enzymes of glycolysis, cytoskeleton proteins)

70

8/12/2019 Outline Ch 08

7/21

2. Peripheral proteins of the cytosolic surface of membranes (spectrins, ankyrins; weakly associated withthe plasma membrane's cytosolic surface)

3. Proteins that are transported to nucleus4. Proteins to be incorporated into peroxisomes, chloroplasts, mitochondria; latter 2 groups made in

cytosol & imported fully formed (posttranslationally)acrossmembrane into appropriateorganelle

II. Why are proteins madeat differentcell sites & how are they identified?- SignalHypothesis;GnterBlbel,David Sabatini& Bernhard Dobberstein (RockefellerU.,early 1970s)A. Suggested & demonstrated that the site of protein synthesis is determined by information (amino acid

sequence) contained in N-terminal portion of protein (first part to emerge from ribosome)1. SecretoryproteinshaveN-terminalsignal sequence thatdirects emerging protein & ribosome to ER2. Signal sequence triggers attachment of protein-making ribosomes to ER & protein movement into

cisternal space through protein-lined, aqueous ER channel as it is being made (cotranslationally)B. Some transport into ER is posttranslational- protein is made totally in cytosol& then imported into ER

1. Goes through same channels as in cotranslational pathway; similar to mechanism of mitochondrial &peroxisomal transport

2. Pathway is used much more heavily in yeast than in mammalian cells for import into ER; yeast cansurvive without cotranslational transport even though they grow more slowly than normal cells

C. Signal hypothesis has been substantiated by a large body of experimental evidence1. Blbel's concept that proteins contain their own "address codes" has been shown to apply in principle to

virtually all types of protein trafficking pathways throughout cell

III. Steps in synthesis of secretory/lysosomal/plant vacuolar protein on membrane-bound ribosomesA. mRNA for secretory/lysosomal/plant vacuolar protein binds to free ribosome (same as those used for

domestic proteins) from pool; these ribosomes are not attached to a cytoplasmic membraneB. N-terminal aminos emerge from ribosome with signal sequence (6-15 hydrophobic amino residues); targets

nascent polypeptide & ribosome for ER1. The signal sequence targets the nascent polypeptide to the ER membrane (a nascent polypeptide is one

in the process of being synthesized & thus is not yet fully assembled)2. Signal sequence leads to compartmentalization of polypeptide within ER lumen3. Signal is usuallyfoundat or nearN-terminus,butoccupiesaninternalposition insomepolypeptides

C. Signal sequence is recognized by signal recognition particle(SRP) as it exits ribosome; SRP inmammalian cells consists of 6 distinct polypeptides & a small RNA molecule (the 7S RNA)1. SRP binds to nascentpolypeptide'ssignal sequence& ribosome (Step 1),temporarily arresting further

synthesis of polypeptideD. Bound SRP serves as tag allowing entire complex (SRP-ribosome-nascent polypeptide) to bind to SRP

receptoron ER cytosolicsurface specifically; this binding occurs through at least 2 distinct interactions1. First distinct interaction is between SRP & SRP receptor2. The other interaction is between ribosome & translocon

E. The translocon is a protein-lined channel embedded in the ER membrane through which the nascentpolypeptide is able to move in its passage from the cytosol to the ER lumen1. Prokaryotic translocon 3D structure was determined by X-ray crystallography & revealed presence of a

pore within translocon in shape of an hourglass

2. The pore had a ring of 6 hydrophobic amino acids situated at its narrowest diameter3. In the inactive (nontranslocating) state, which was the state in which the structure was crystallized,the opening in the pore ring is plugged by a short helix

4. This plug is proposed to seal the channel, preventing the unwanted passage of calcium & other ionsbetween the cytosol & the ER lumen

F. Once the SRP-ribosome-nascent chain complex binds to the ER membrane (Step 2), the SRP is releasedfrom its ER receptor & the ribosome is attached to translocon's cytosolic end & then1. The nascent polypeptide's signal sequence is inserted into the translocon's narrow aqueous channel

(Step 3)

71

8/12/2019 Outline Ch 08

8/21

2. It is proposed that contact of signal sequence with the translocon interior leads to displacement of theplug & opening of the passageway

G. Growing polypeptide is then translocated through hydrophobic pore ring & into ER lumen (Step 4)1. The pore ring seen in crystal structure has a diameter (5-8 ), considerably smaller than that of a helica

polypeptide chain, so it is presumed that pore expands as nascent chain traverses channel2. Expansion is possible because the residues that make up the ring are situated on different helices

H. Upon translation termination & completed polypeptide's passage through translocon, the membrane-boundribosomeis released from ER membrane; helical plugis then reinsertedinto translocon channel

IV. GTP is involved in secretory protein synthesis - several steps are regulated by its binding or hydrolysisA. G-proteins (GTP-binding proteins) play key regulatory roles in many different cellular processes

1. G-proteins exist in at least 2 alternate conformations: active GTP-bound & inactive GDP-bound form;the 2 conformations have different abilities to bind other proteins

2. Thus, G-proteins act like molecular switches turning specific processes on and off; the GTP-bindingproteintypically turns process on & hydrolysis of bound GTP to GDP turns process off

3. Also GTP-binding-proteins generally require accessory proteins to carry out their functionB. SRP & SRP receptor (2 major interactants in the above process) are both G proteins that interact with one

another in their GTP-bound states (unusual)1. Hydrolysis of GTP bound to these two proteins occurs between steps 2 & 3 & triggers the release of the

signal sequence by the SRP & its insertion into the translocon

The Endoplasmic eticulum !E"# (rocessin& of Newly Synthesi,ed (roteins in

the Endoplasmic eticulum

I. As it enters RER cisterna,a nascent polypeptide is acted upon by a variety of enzymes located within either themembraneor lumen of the RERA. Signal peptide on N-terminus of nascent polypeptide is removed from most of the nascent proteins by a

proteolytic enzyme, the signal peptidaseB. Carbohydrates are added to nascent protein by enzyme oligosaccharyltransferase

1. Both signal peptidase & oligosaccharyltransferase are integral membrane proteins residing in closeproximity to translocon

2. Both enzymes act on the nascent proteins as they enter the ER lumen

II. The RER is a major protein processing plantA. To meet its obligations, RER lumen is packed with molecular chaperonesthat recognize & bind to

unfolded or misfolded proteins& give themopportunityto attain their correct (native) 3D structureB. The ER lumen also contains a number of protein-processing enzymes, like protein disulfide isomerase

(PDI)1. Proteins enter ER lumen with their cysteine residues in the reduced (SH) state, but theyleave the

compartment with many of these residues joined to one another as oxidized disulfides (S-S)2. The formation (& rearrangement) of disulfide bonds is catalyzed by PDI3. Disulfide bonds play an important role in maintaining the stability of proteins that are present at the

extracellular surface of the plasma membrane or secreted into the extracellular space

III. The ER is ideally constructed for its role as a port of entry for the biosynthetic pathway of the cellA. Its membrane provides a large surface area to which many ribosomes can attach (an estimated 13

million/liver cell)B. ER cisternae lumen provides local environment that favors protein folding & assemblyC. ER cisternae lumen also provides a compartment in which secretory, lysosomal & plant-cell vacuolar

proteins can be segregated from other newly made proteins1. This segregation of newly made proteins in ER cisternae removes them from cytosol

72

8/12/2019 Outline Ch 08

9/21

2. It also allows them to be modified & dispatched toward their ultimate destination, whether outside thecell or within one of the cytoplasm's membranous organelles

The Endoplasmic eticulum !E"# Synthesis of Inte&ral )embrane (roteins on)embrane*$ound ibosomes

I. Integral membrane proteins (other than those of mitochondria & chloroplasts) are also synthesized onmembrane-bound ribosomes of ERA. These membrane proteins are translocated into ER membrane as they are synthesized (cotranslationally)

using the same machinery used for synthesis of secretory & lysosomal proteins1. Unlikesoluble secretory &lysosomal proteins,however, which pass entirely through ER membrane

during translocation, integral proteins contain 1 hydrophobic transmembrane segments2. These hydrophobic transmembrane segments are shunted directly from the translocon channel into the

lipid bilayer how can this take place?B. X-ray crystallographic studies of translocon showed translocon to have a clam-shaped conformation with a

groove or seam along one side of the wall where the channel might open & close1. As protein moves through translocon, it is thought that lateral gate in channel continually opens &

closes; allows each nascent polypeptidesegment topartition itself according to solubility properties

2. Each segment may stay in the aqueous compartment within translocon channel or move into thesurrounding hydrophobic lipid bilayer core3. The segments of nascent polypeptide that are sufficiently hydrophobic will spontaneously dissolve into

lipid bilayer & ultimately become transmembrane integral membrane protein segmentsC. This idea has received strong support from in vitrostudy in which translocons were given the chance to

translocate custom-designed nascent proteins containing test segments of varying hydrophobicity1. The more hydrophobic the test segment, the greater the likelihood that it will pass through the wall of

the translocon & become integrated as a transmembrane segment of the bilayer

II. Single-spanning membrane proteins can have an orientation with their N-terminus facing either the cytosol orthe ER lumen (& eventually the extracellular space)A. The most common determinant of membrane protein alignment is the presence of positively-charged amino

acid residues flanking the cytosolic end of a transmembrane segment

B. During membrane protein synthesis, the inner lining of translocon is thought to orient the nascentpolypeptide so that the more positive end faces the cytosol

III. In multispanning proteins, sequential transmembrane segments typically have opposite orientationsA. For these proteins, their arrangement within the membrane is determined by the orientation in which the

first transmembrane segment is inserted1. Once that has been determined, every other transmembrane segment has to be rotated 180 before it can

exit the transloconB. Studies performed with purified components in cell-free systems suggest that a translocon, by itself, is

capable of properly orienting transmembrane segmentsC. It appears that translocon is more than a simple passageway through ER membrane; it is a complex machin

that can recognize various signal sequences & perform complex mechanical activities

The Endoplasmic eticulum !E"# )embrane $iosynthesis in the E

I. Membranes thought to arise only from pre-existing membranes (not de novo[new entities from pools ofproteins & lipids])A. Membranes grow as newly made proteins & lipids are inserted into existing membranes in ER; each

compartment has unique membranes1. Membrane components move from ER to virtually every other cell compartment

73

8/12/2019 Outline Ch 08

10/21

2. As membrane moves from compartment to compartment in cell, its proteins & lipids are modified byenzymes residing in the cell's various organelles

3. Modifications contribute to giving each membrane compartment a unique composition & distinctidentity

4. These modifications are done by the same enzymes that modify secretory proteins that are free in the ERlumen

B. Cell membranes are asymmetric; the 2 phospholipid layers (leaflets) have different compositions1. Asymmetry is initially established in ER as lipids & proteins are inserted preferentially into one layer othe other

2. Asymmetry is maintained while membrane passes through cell by budding from one compartment &fusing to the next

3. Thus, components situated at cytosolic surface of ER membrane can be identified on cytosolic surfacesof transport vesicles, Golgi cisternae & internal (cytoplasmic) surface of plasma membrane

4. Similarly, components situated at luminal surface of ER membrane naintain their orientation & are founat the external (exoplasmic) surface of the plasma membrane

5. In many ways, including high calcium concentration & abundance of proteins with disulfide bonds &carbohydrate chains,a. The ER lumen (as well as other compartments of the secretory pathway) is a lot like the extracellular

space

II. Synthesis of membrane lipidsA. Most membrane lipids are produced entirely in ER membrane with following exceptions:

1. Sphingomyelin&glycolipids, the synthesis of which starts in ER & is completed in Golgi complex2. Some unique mitochondrial/chloroplast membrane lipids (made by enzymes in those membranes)

B. Phospholipids are made by integral ER membrane enzymes whose active sites face cytosol1. Newly synthesizedphospholipids are inserted into the outer(cytoplasmic)leafletof ERmembrane2. Some of the lipids move to inner leaflet aided by flippases (actively translocate them across bilayer)3. Lipids are carried from ER to Golgi complex & plasma membrane as part of bilayers making up

transport vesicle wallsC. Membranes of different organelles have markedly different lipid composition (changes made as membrane

flows through cell) - what factors contribute to these changes?

1. Conversion of one type of phospholipid to another - most organelles have enzymes that modify lipidsalready present in membrane (example phosphatidylserine to phosphatidylcholine)2. Asmembranesbud,some phospholipids preferentiallyincluded in forming vesicle, others excluded3. Phospholipid-transfer proteinsmove specific phospholipids between membrane compartments throug

aqueouscytosol&maymove themfromERtootherorganelles(mitochondria,chloroplasts)

The Endoplasmic eticulum !E"# 'lycosylation in the ou&h Endoplasmic

eticulum

I. Most proteins made on RER are glycosylated & thus become glycoproteins, whether integral proteins ofmembrane, soluble lysosomal or vacuolar enzymes or parts of ECMA. Carbohydrate groups have key roles in function of many glycoproteins (e. g., binding sites in their

interactionswithothermacromolecules as occurs during many cellular processes)

1. They alsoaidin properfoldingof the proteintowhichtheyare attached2. Sugar sequences that comprise glycoprotein oligosaccharides are highly specific3. Sugar sequences from purified glycoprotein are consistent & predictable - how determined?

B. Howisoligosaccharidesugarsequenceassembled? catalyzedbya family ofmembrane-boundenzymes(glycosyltransferases)1. Each of these glycosyltransferases transfers a specific monosaccharide from a nucleotide sugar2. Donor is always a nucleotide sugar - GDP-mannose, GDP-fucose, UDP-galactose, UDP-N-

acetylglucosamine; acceptor of transferred sugar is growing end of carbohydrate chain

74

8/12/2019 Outline Ch 08

11/21

3. Sequence of sugar transfer during oligosaccharide assembly depends on the sequence of action ofglycosyltransferases participating in process

4. Glycosyltransferase sequence, in turn, depends on the location of specific enzymes within the varioussecretory pathway membranes

5. Thus, sugar arrangement in oligosaccharide chains of a glycoprotein depends on the spatial localizationof certain enzymes in this assembly line

II. Carbohydratechainsareattachedto proteinby N-linkages(asparagineN atom) or O-linkages (to serine orthreonine O or collagen hydroxylysine residue) of both soluble & integral membrane proteinsA. These oligosaccharides differ in average size, sugar composition & path of synthesis & also share

properties like their high specificityB. N-linked basal (core) chain segment is assembled on lipid carrier not protein; then transferred as a block to

specific asparagine residues of polypeptide as it enters RER by oligosaccharyltransferase1. Lipid carrier is dolichol phosphate; embedded in membrane (hydrophobic molecule built from >20

isoprene units) & sugars are added one at a time by membrane-bound glycosyltransferases2. This part of glycosylation process is essentially invariant3. In mammalian cells, it starts with transfer ofN-acetylglucosamine 1-phosphate & then transfer of

anotherN-acetylglucosamine, then 9 mannose & 3 glucose units in a precise pattern4. This preassembled block of 14 sugars is then transferred by oligosaccharyltransferase from dolichol

phosphate to nascent polypeptide as it is being translocated into ER lumen

III. Mutationsthat lead to total absence ofN-glycosylationcause death of embryos prior to implantation;A. Mutations leading to partial glycosylation pathway disruption in ER also cause serious inherited disorders

affecting nearly every organ systemB. These diseases are called Congenital Diseases of Glycosylation (CDGs) & they are usually identified

through blood tests that detect abnormal glycosylation of serum proteinsC. Example: One of these diseases, CDG1b can be managed through a remarkably simple treatment

1. It results from deficiency of the enzyme phosphomannose isomerase (catalyzes conversion of fructose-6-phosphate to mannose-6-phosphate)

2. Its reaction is a crucial reaction in the pathway that makes mannose available for incorporation intooligosaccharides

3. The disease can be managed by giving patients oral supplements of mannose; first tested in boy whowas dying from uncontrolled gastrointestinal bleeding (a usual complication of the disease)4. Within months of taking mannose supplements, the child was living a normal life

IV. Shortly after it is transferred to the nascent polypeptide, the oligosaccharide chain undergoes a gradualprocess of modificationA. This modification begins in the ERwiththeenzymaticremovalof2 of the 3terminalglucose residuesby

glucosidasesB. This sets the stage for an important event in a newly made glycoprotein's life

1. During this stage, the glycoprotein is screened by a system of quality control that determines whether onot it is fit to move to the next compartment of the biosynthetic pathway

2. The screening process begins with each glycoprotein, which at this stage contains a single remainingglucose, binding to an ER chaperone (calnexin or calreticulin)

3. Removal of remaining glucose by glucosidase II leads to release of glycoprotein from chaperoneC. Iffoldingisincompleteorifproteinismisfolded,it is recognized & boundbyconformation-sensingenzyme

(called GT)1. If GT binds to the glycoprotein, it adds a single glucose back to one of the mannose residues at the

exposed end of the recently trimmed oligosaccharide2. GT recognizes incompletely folded or misfolded proteins because they display exposed hydrophobic

residues that are absent from properly folded proteins

75

8/12/2019 Outline Ch 08

12/21

3. Once the glucose residue is added, the tagged glycoprotein is recognized by the same chaperones givingit another chance to fold properly

4. After some time with chaperone, the added glucose is removed & conformation-sensing GT checksprotein again to see if it has achieved its proper 3D structure (is it partially unfolded or misfolded?)

5. If 3D structure is right, protein continues on its way; if not, glucose is added & process repeats untileventually, the glycoprotein has folded correctly or it remains misfolded & is destroyed

6. Studies suggest the "decision" to destroy the defective protein is governed by a slow acting enzyme in Ea. It trims a mannose residue from an exposed end of the oligosaccharide of a protein that has been in thER for an extended period

b. Once one or more of these mannose residues has been removed, the protein can no longer be recycled&, instead, is sentenced to degradation

The Endoplasmic eticulum !E"# )echanisms That Ensure Destruction of

)isfolded (roteins

I. MisfoldedproteinsarenotdestroyedinER, butare insteadtransportedinto cytosol by dislocationA. It remains unclear whether misfolded proteins are dislocated back into cytosol through translocons that

brought them into ER or by way of a separate dislocation channel of uncertain identityB. Once in cytosol,misfolded proteinsare destroyed in proteasomes,which are protein-degrading machines;

this process ensures that aberrant proteins are not transported to other parts of cell1. But this can have negative consequences; in most cases of cystic fibrosis, the plasma membrane of

epithelial cells is lacking the abnormal protein encoded by the cystic fibrosis gene2. In these cases, the mutant protein is destroyed by the quality control process & thus fails to reach the

cell surface

II. Sometimes, misfolded proteins can be generated in ER at a rate faster than they can be exported to thecytoplasmA. The accumulation of misfolded proteins, which is a potentially lethal situation, triggers a comprehensive

"plan of action" within the cells known as the unfolded protein response (UPR)B. TheER containssensors that monitorthe concentrationof unfoldedor misfolded proteins in ER lumeC. The prevailing model suggests that the sensors are normally kept in an inactive state by molecular

chaperones, particularly BiP1. If circumstances lead to an accumulation of misfolded proteins, the BiP molecules in the ER lumenbecome "tied up" as a result of their interaction with the misfolded proteins

2. This renders them (the BiP molecules) incapable of inhibiting the sensors; activation of the sensorsleads to a multitude of signals that are transmitted into both the nucleus & cytosol

3. This results in the expression of hundreds of different genes whose encoded proteins have thepotential to alleviate stressful conditions within the ER

D. Among the genes expressed are genes that encode:1. ER-based molecular chaperones that can help proteins reach the native state2. Proteins involved in the transport of the proteins out of the ER3. Proteins involved in the selective destruction of abnormal proteins as described above

E. The UPR is more than cell-survival mechanism; it includes the activation of a cell-death pathway1. It is presumed that the UPR gives the cell an opportunity to relieve itself of the stressful conditions

2. If these corrective measures are unsuccessful, the cell-death pathway is triggered & cell is destroyed

From the E to the 'ol&i Comple-# The First Step in .esicular Transport

I. The exit sites of RER cisternae are typically smooth-surfaced (devoid of ribosomes) & serve as places wherethe first transport vesicles in biosynthetic pathway are formed

II. Trip from ER to Golgi has been visualized in living cells by tagging secretory proteins with green fluorescentprotein (GFP)

76

8/12/2019 Outline Ch 08

13/21

A. After budding from ER membrane, transport vesicles are seen to fuse to each other to form larger vesicles& interconnected tubules in region between ER & Golgi complex

B. This region is called ERGIC (endoplasmicreticulum Golgi intermediate compartment) & the vesicular-tubularclusters that form there are called VTCs

C. Once formed, VTCs move farther away from the ER toward Golgi complex; other studies indicate that thismovement occurs along tracks composed of microtubules

The 'ol&i Comple-

I. Discovered by Camillo Golgi (Italian biologist, 1898) inventor of new types of staining procedures that hehoped might reveal the organization of nerve cells within the central nervous systemA. One stainused solutionof silver nitrateapplied to tissue that had been soaked in osmium& bichromate

1. Applied stain for several days to cerebellum nerve cells & saw darkly staining reticular network near thcell nucleus; he got the Nobel Prize in part for this discovery in 1906

2. Later seen in other cell types & named Golgi complex; some believed it existed in living cells, othersthought it was an artifact (artificial structure formed during preparation for microscopy)

3. For decades, the reality of its existence was the center of a controversyB. Existence confirmed beyond a reasonable doubt when it was clearly identified in unfixed, freeze-fractured

cells; it was no artifact

II. Characteristic morphology - flattened, disk-like membranous cisternae with dilated rims & associated vesicles& tubules (smooth membranes so found with smooth microsomes)A. Cisternae (typically 0.5 - 1.0 m dia) arranged in orderly stack like pancakes; curved resembling a shallow

bowl; individual Golgi stacks often interconnected to form ribbonlike complex1. In plants, a single Golgi stack is sometimes called dictyosome

B. Usually

8/12/2019 Outline Ch 08

14/21

C. Golgi complex composition is not uniform from cis- to trans-end; polarized; differences in composition ofmembrane compartments (polarization) reflects primary processing plant role1. Newly synthesized membrane proteins (also secretory & lysosomal proteins) leave the ER & enter the

Golgi complex at its cis-face & then pass across the stack to the transfaceD. As they move along the stack, proteins originally synthesized in RER are sequentially modified in specific

ways; for example:

1. Part of the protein's length may be trimmed by proteolytic enzymes2. Aminoacids may be modified(hydroxylationof lysine & proline residues of a collagen molecule)3. The protein's carbohydrates are modified by a series of stepwise enzymatic reactions

IV. Glycosylation in Golgi complex - synthesis site of most of cells complex polysaccharides (animal ECMGAGs; plant cell wall pectins & hemicellulose); key role in glycoprotein/glycolipid CHO assemblyA. In ER, glucose residues had just been removed (see above) from the ends of core oligosaccharide ofN-

linked CHO chains1. As newly synthesized soluble & membrane glycoproteins pass though cis &medialGolgi cisternae,

most of the mannose residues are also removed from the core oligosaccharides2. Other sugars are added sequentially by various glycosyltransferases to produce a variety of different

oligosaccharidesB. In Golgi, as in RER, sequences in which sugars are inserted into oligosaccharides is determined by spatial

arrangement of specific glycosyltransferases that contact new proteins as they pass through1. Sialyltransferase (puts sialic acid at chain terminal position in animal cells) is found in transend of Golg

stack; expected if new glycoproteins were continually moving toward this part of organelle2. In ER, a single core oligosaccharide is assembled; in Golgi complex, glycosylation steps can be quite

varied, producing carbohydrate domains of remarkable sequence diversity3. Proteins in RER lack sugars that are normally added in medial & transGolgi cisternae

C. Unlike N-linked oligosaccharides, whose synthesis starts in ER, those attached to proteins by O-linkages arassembled wholly within Golgi complex

V. Vesicular transport within Golgi; how do materials move through Golgi? > 2 contrasting theoriesA. Cisternal maturation model (up to mid-1980s) it was accepted that cisternae were transient structures;

form at cisface by ER/ERGIC vesicle fusion, travel totransface & altered along the way

1. Cisternae mature & change in composition as they move through Golgi complex; each cisterna maturesinto next cisterna along stack (origin of name)2. Each cisterna was thought to physically move from the cisto the transend of the stack, changing in

composition as it progressedB. New model favored (mid-1980s until late-1990s) cisternae of Golgi stack remain in place as stable

compartments held together by protein scaffold; known as the Vesicular Transport Model1. Cargo (secretory, lysosomal, membrane proteins) is shuttled through Golgi stack from CGN to TGN in

vesicles that bud from one compartment & fuse with neighboring one farther along stack

VI. Acceptance of Vesicular Transport Model based largely on the following observations:A. Eachofthe various Golgi cisternae ofstack has distinct resident enzyme population;howcould various

cisternae have such different properties if each gave rise to next in line as stated by other model?B. Large numbers of vesicles are seen in electron micrographs to bud from rims of Golgi cisternae - James

Rothman, et al. (Stanford, 1983)1. Using cell-free preparations of Golgi membranes, they showed that transport vesicles could bud from

one Golgi cisterna & fuse with another Golgi cisterna in vitro2. Formed basis for hypothesis suggesting that inside cell, cargo-bearing vesicles budded from cis-

cisternae & fused with cisternae derived from a more trans positionin stack

VII. Both models still have proponents, but consensus has shifted in past few years back to cisternal maturationmodel; several major reasons summarized below:

78

8/12/2019 Outline Ch 08

15/21

A. Cisternal maturation (CM) model envisions a highly dynamic Golgi complex in which major elements oforganelle, the cisternae, are continually being formed at the cisface & dispersed at the transface1. According to this view, the very existence of the Golgi complex itself depends on the continual influx o

transport carriers from the ER & ERGIC2. As CM model says, when transport carrier formation from ER is blocked either by cell treatment with

specific drugs or use of temperature-sensitive mutants, Golgi complex simply disappears

3. When the drugs are removed or the mutant cells are returned to the permissive temperature, the Golgicomplex rapidly reassembles as ER-to-Golgi transport is renewedB. New evidence for CM model - certain materials that are produced in ER & then travel through Golgi

complexcan be shownto stay in Golgi cisternae& neverappearwithinGolgi-associated transport vesicles1. Example: fibroblast studies large complexes of procollagen molecules (extracellular collagen

precursors) move from ciscisternae to transcisternae without ever leaving the cisternal lumenC. Until mid-1990s, it was assumed that transport vesicles always moved in forward (anterograde) direction,

from cisorigin to transdestination, but new evidence says that1. Some move in backward (retrograde) direction from transdonor to cisacceptor membrane

VIII. Revised cisternal maturation model acknowledges a role for transport vesicles, which have been clearlyshown to bud from Golgi membranesA. In this model, transport vesicles do not shuttle cargo in anterograde direction, but instead carry resident

Golgi enzymes in retrograde direction1. This model of intra-Golgi transport is supported by electron micrographs showing ultra-thin sections of

cultured mammalian cells that were cut from a frozen block2. Frozen sections were treated with antibodies that were linked to gold particles prior to examination in

EM; the antibodies were made against a cargo protein (the viral protein VSVG protein)3. VSVG molecules were present within cisternae, but absent from nearby vesicles, suggesting that cargo

carried inanterograde directionwithin maturingcisternae butnot in smalltransport vesiclesB. In another experiment, treated gold-labeled antibodies that bind to a Golgi resident protein (the enzyme

mannosidase II) > it was found in both the cisternae & associated vesicles1. This strongly supports the proposal that these vesicles are utilized to carry Golgi-resident enzymes in a

retrograde directionC. The revised cisternal maturation model explains how different Golgi cisternae in a stack can have a unique

identity1. The enzyme mannosidase II removes mannose residues from oligosaccharides & is mostly restricted to

medialcisternae2. It can be recycled backward in transport vesicles as each cisterna moves toward transend of stack

D. Some prominent researchers still argue, based on other experimental results, that cargo can be carried bytransport vesicles between Golgi cisternae in an anterograde direction, so matter is not yet settled

The Types of .esicle Transport and Their Function# $ac%&round Information

I. Materials carried between membrane compartments by vesicles or other types of membrane-bound carriers,which bud from donor membranes & fuse with acceptor membranes

A. Most budding vesicles covered on cytosolic surface by fuzzy, electron-dense layer1. The dark-staining layer consists of a protein coat formed from soluble proteins that assemble on thedonor membrane cytosolic surface at sites where budding takes place

2. Each coated bud pinches off to form a coated vesicle; assembly is initiated by the activation of asmall G protein that is specifically recruited to the site

3. Vesicles of similar size & structure can be formed in cell-free systemsB. Protein coats have at least two distinct functions:

1. They act as a mechanical device that causes the membrane to curve & form a budding vesicle2. Theyprovidea mechanismfor selectingcomponents(&thus soluble cargo) to be carried by vesicle

79

8/12/2019 Outline Ch 08

16/21

C. Components selected for transport can include:1. Cargo to be transported (secretory, lysosomal, & membrane proteins)2. Machinery required to target & dock the vesicle to an acceptor membrane

D. In the two best-understood cases, the vesicle coat is composed of 2 distinct protein layers1. An outer cage or scaffolding that forms the framework for the coat &2. An inner layer of adaptors that serves primarily to bind the vesicle's cargo

a. The adaptors are able to select specific cargo molecules by virtue of their specific affinity for thecytosolic "tails" of integral proteins that reside in the donor membrane

II. Several distinct classes of coated vesicles have been identified - distinguished by the proteins that make up thecoat, their appearance in EM, & their role in cell trafficking; three are the best-studied:A. COPII-coated vesicles - move materials forward from ER to ERGIC (intermediate compartment between

ER & Golgi) & Golgi complex; COP is acronym for coat proteinsB. COPI-coated vesicles - move materials in retrograde direction from ERGIC & Golgi stack backward toward

ER1. Also thought to transport materials through Golgi from cisto transface2. May play role in trafficking from ER to Golgi, from TGN to cell membrane, between compartments of

endocytic pathwayC. Clathrin-coated vesicles - move materials from TGN to endosomes, lysosomes & plant vacuoles

1. Alsomove materialsfrom plasmamembraneto cytoplasmiccompartmentsalongendocyticpathway2. Also implicated in trafficking from endosomes & lysosomes

CO(II*Coated .esicles# Transportin& Car&o from the E to the 'ol&i Comple-

I. COPII-coated vesicles are the most recently discovered & mediate the first leg of journey through thebiosynthetic pathway from ER to ERGIC & CGNA. COPII coat containsa number of proteinsfirstfoundin mutantyeast cells that could not transport materials

fromERtoGolgi;homologousproteinsfound incoatsof vesicles budding from mammalian cell ERB. Antibodies to COPII-coat proteins block vesicle budding from ER membranes but have no effect on

movement of cargo at other stages in the secretory pathway

II. COPII-coats select&concentratecertaincomponentsfor transport in vesiclesA. Certain ER integral membrane proteins are selectively captured because they interact specifically with

COPII proteins of vesicle coat; several types of membrane proteins are included in this group:1. Enzymesthat act at laterstages of biosyntheticpathway, like glycosyltransferases of Golgi complex2. Membrane proteins involved in docking & fusion of the vesicle with the target compartment3. Membrane proteins that bind soluble cargo (secretory proteins), e. g., membrane protein, ERGIC-53, th

binds to mannose residues found on oligosaccharides of certain secretory proteins in ERB. Example: ERGIC-53 mutations have been linked to an inherited bleeding disorder; people with the disorde

fail to secrete certain coagulation factors that promote blood clotting

III. Interaction between membrane proteins (like ERGIC-53) & the COPII-coat is mediated by signal sequences ithe cytosolic tails of the membrane proteins

A. ERGIC-53, for example, is recognized by 2 neighboring phenylalanines in its cytosolic tailB. Other types of soluble cargo are not selected at this stage & are present at the same concentration in thebudding vesicle as in ER lumen1. Proteins that become enclosed in vesicles but are not specifically selected for inclusion are said to move

by bulk flow2. Some of the integral ER membrane proteins may also become trapped in budding vesicles & transporte

through secretory pathway to plasma membrane by bulk flow

80

8/12/2019 Outline Ch 08

17/21

IV. Among COPII coat proteins is a small G protein (Sar1); it is recruited specifically to ER membrane; like otheG proteins Sar1 plays regulatory role, here, it starts vesicle formation & regulates vesicle coat assemblyA. First, Sar1 is recruited to the ER membrane in the GDP-bound form & is induced to exchange its GDP for

GTP molecule (Step 1)B. Upon binding GTP, Sar1 undergoes a conformational change that causes its N-terminal helix to insert

itself into the cytosolic leaflet of the ER bilayer (Step 2)

1. This event has been shown to bend the lipid bilayer, which is an important step in the conversion of aflattened membrane into a spherical vesicle

2. Membrane bending is probably aided by a change in packing of the lipids that make up the 2 leaflets ofthe bilayer

C. In Step 3, Sar1GTP has recruited 2 additional polypeptides of the COPII coat, Sec23 & Sec24, which bindas a "banana-shaped" dimer1. Because of its curved shape, the Sec23-Sec24 dimer provides additional pressure on the membrane

surface to help it further bend into a curved bud2. Sec24alsofunctionsas primaryadaptorproteinof COPIIcoat that interacts specifically with the ER

export signals in membrane protein cytosolic tails that are destined to traffic on to Golgi complexD. In step 4, the remaining subunits of the COPII coat, Sec13 & Sec31, bind to the membrane to form the oute

structural cage of the protein coat

1. The Sec13-Sec31 cage assembles into a relatively simple lattice in which each vertex is formed by theconvergence of 4 Sec13-Sec31 legs2. A certain degree of flexibility is built into the interface between the Sec13-Sec31 subunits that allow

them to form cages of varying diameter, thus accommodating vesicles of varying size3. Once the entire COPII coat has assembled, the bud is separated from the ER membrane in the form of a

COPII-coated vesicleE. Before the coated vesicle can fuse with a target membrane, the protein coat must be disassembled and its

components released into the cytosol1. Disassembly is triggered by hydrolysis of the bound GTP to produce a Sar1-GDP subunit, which has

decreased affinity for the vesicle membrane2. Dissociation of Sar1-GDP from the membrane is followed by the release of the other COPII subunits

CO(I*Coated .esicles# Transportin& Escaped (roteins $ac% to the EI. COPI-coated vesicles were first identified in experiments where cells were treated with GTP analogues

(molecules with structures similar to GTP) that cannot be hydrolyzed (unlike GTP)A. In the presence of these analogues, COPI-coated vesicles accumulated within the cell & could be isolated

from homogenized cells by density gradient centrifugation1. They accumulate in presence of analogue because (like COPII coat) their coat contains a small GTP-

binding protein (ARF1), whose bound GTP must be hydrolyzed before the coat can disassemble2. ARF1 (adenosylationribosefactor) is 1 of 8 distinct proteins to make up complete COPI coat

B. COPI-coated vesicles have been most clearly implicated in the retrograde transport of proteins including thmovement of:1. Golgi-resident enzymes in a trans-to-cisdirection (like mannosidase II)2. ER-resident enzymes from the ERGIC & the Golgi complex back to the ER

C. Whether or not COPI-coated vesicles are involved in anterograde and/or retrograde transport between Golgcisternae remains a matter of controversy

II. Retaining & retrieving resident ER proteinsA. Questions about the process of retention & retrieval of resident ER proteins

1. If vesicles continually bud from membrane compartments, how does each compartment retain its uniqucomposition?

2. What determineswhethera particularER membrane proteinstays inER or goeson to Golgicomplex?B. Studies suggest proteins are maintained in an organelle by a combination of 2 mechanisms:

81

8/12/2019 Outline Ch 08

18/21

1. Retention of resident molecules that are excluded from transport vesiclesa. Retention may be based primarily on the physical properties of the proteinb. For example, soluble proteins that are part of large complexes or membrane proteins with short

transmembrane domains are not likely to enter a transport vesicle2. Retrieval of "escaped" molecules back to the compartment in which they normally reside

III. Retrieval of escaped proteins is better understood - proteins that normally reside in ER (in lumen & themembrane) have short amino acid sequences at C-terminus that serve as retrieval signalsA. This ensures their return to ER if they are carried forward accidentally in transport vesicle to ERGIC or

Golgi complexB. The retrieval of "escaped" ER proteins from these compartments is accomplished by specific receptors that

capture the molecules & return them to the ER in COPI-coated vesiclesC. Soluble proteins of ER lumen (protein disulfide isomerase & molecular chaperones that facilitate folding)

typically possess the retrieval signal "lys-asp-glu-leu" [KDEL in single letter nomenclature]1. Soluble ER proteins with KDEL signal are recognized & returned to the ER bound by an integral

membrane protein, the KDEL receptor, that shuttles between the cisGolgi & the ER compartmentsa. The receptor's cytosolic tail binds to COPI coat, ensuring its return to ER

2. If KDEL sequence is deleted from ER protein, the ER proteins are not recovered & brought back to theER compartment, but instead they are carried forward through the Golgi complex

3. If a scientist engineers the gene for a lysosomal or secretory protein in the cell to have an added KDELC-terminus, the protein is returned to ER rather than being sent to its proper destination

D. Membrane proteins that reside in the ER also have a retrieval signal that binds to the COPI coat, facilitatingtheir return to the ER1. The most common retrieval sequences for ER membrane proteins involve two closely linked basic

residues, most commonly KKXX (where K is lysine & X is any residue)E. Each biosynthetic pathway compartment may have its own unique retrieval signals; this explains the

maintenance of unique protein complements in each one despite constant in/out vesicle movement

$eyond the 'ol&i Comple-# Sortin& (roteins at the T'N

I. How does particular protein synthesized in ER get targeted toward particular cellular destination?A. Cell must be able to distinguish among the various proteins it manufactures example: pancreatic cell

1. Must segregatenewly made digestive enzymes (secreted into duct), fromnewlymadecell-adhesionmolecules(ultimately residein plasma membrane),fromlysosomal enzymesdestinedfor lysosomes

2. So the cell sorts proteins destined for different sites into different vesicles, determining destinationB. Protein sorting occurs in the last of the Golgi compartments, the transGolgi network (TGN), which

functions as a major branch point in the movement of materials along the secretory pathway1. The TGN is the site of assembly of clathrin-coated vesicles2. Clathrin coats mediate cargo sorting at TGN & clathrin-coated vesicles carry hydrolytic enzymes &

membrane proteins from there to endosomes, lysosomes & plant vacuoles

II. Lysosomal protein sorting & transport - made on membrane-bound RER ribosomes, carried to cis Golgicisternaewith other protein types; this is the best understood post-Golgi pathway (for lysosomal enzymes)

A. Once in Golgi cisternae,solublelysosomal enzymes recognized by enzymes catalyzing 2-step addition ofphosphategrouptocertainN-linkedCHO chain mannosesugars1. Unlike other glycoproteins sorted at the TGN, lysosomal enzymes possess phosphorylated mannose

residues, which act as recognition signals2. This mechanism of protein sorting was discovered through studies on human cells that lacked one of th

enzymes involved in phosphate additionB. Lysosomal enzymes with mannose 6-phosphate signal are recognized & captured by mannose-6-phosphate

receptors (MPRs; integral membrane proteins that span the Golgi membranes)C. Lysosomalenzymesare transportedfrom TGN in clathrin-coated vesicles; coats of the vesicles contain:

82

8/12/2019 Outline Ch 08

19/21

1. An outer honeycomblikelatticecomposed ofthe protein clathrin, which forms a structural scaffold2. An inner shell made of proteinadaptors that coverthe vesicle membranesurface facingthecytosol; in

molecular biology, an adaptor is a molecule that physically links 2 different types of materialsD. Lysosomal enzymes are escorted from the TGN by a family of adaptor proteins called GGAs

1. Each GGA molecule has several domains, each capable of grasping a different protein involved invesicle formation

2. The outer ends of GGA adaptors bind to clathrin molecules, holding the clathrin scaffolding onto thesurface of the vesicle3. On their inner surface, GGA adaptors bind to sorting signals in the cytosolic tails of the mannose 6-

phosphate receptors4. The MPRs, in turn, bind to soluble lysosomal enzymes within the vesicle lumen5. As a result of these interactions with GGA adaptors, MPRs in TGN membrane & lysosomal enzymes

within TGN lumen become concentrated into clathrin-coated vesiclesE. As with COPI/COPII vesicle formation, clathrin-coated vesicle production starts with recruitment to the

membraneof smallGTP-bindingprotein(ARF1),which setsthestage forbindingof othercoatproteinsF. Once the vesicle has budded from the TGN, the clathrin coat is lost & the uncoated vesicle proceeds to its

destination, which may be an endosome, lysosome or plant vacuoleG. Once the vesicle reaches its destination organelle, the MPRs dissociate from the lysosomal enzymes &

return to the TGN for another round of lysosomal enzyme transport

$eyond the 'ol&i Comple-# Sortin& and Transport of Non*/ysosomal (roteins

I. Membrane proteins destined for plasma membrane & secretory proteins destined for export from the cell arealso transported from TGN, but the mechanisms are poorly understoodA. Recent model membranous carriers are produced as the TGN fragments into vesicles & tubules of variou

sizes; this fits with cisternal maturation model1. Cisternal maturation model suggests that Golgi complex cisternae move continually toward TGN, wher

they would have to disperse to allow continued maturation of Golgi stack2. Proteins that are discharged from the cell by a process of regulated secretion (digestive enzymes,

hormones) are thought to form selective aggregates3. These aggregates eventually become contained in large, densely packed secretory granules & are

apparently trapped as immature secretory granules bud from rims oftrans

Golgi cisternae & TGN4. In some cells, long tubules are seen to be pulled out of the TGN by motor proteins that operate alongmicrotubular tracksa. These tubules are then split into a number of vesicles or granules by membrane fisiionb. Once they have departed from the TGN, the contents of the secretory granules become more

concentrated4. Eventually, the mature granules are stored in the cytoplasm until their contents are released after

stimulation of the cell by a hormone or nerve impulse

II. The targeted delivery of integral proteins to the plasma membrane appears to be based largely on sortingsignals in the cytoplasmic domains of the membrane proteinsA. In polarized cells, membrane proteins destined to reside in the apical portions of the plasma membrane

contain different sorting signals from those destined for the lateral or basal portion

B. Plasma membranes of nonpolarized cells (fibroblasts, white blood cells) may not require special sortingsignals1. Such proteins may simply be carried from the TGN to the cell surface in vesicles of the constitutive

secretory pathway

$eyond the 'ol&i Comple-# Tar&etin& .esicles to a (articular Compartment#

$ac%&round

I. Vesicle fusion requires specific interactions between different membranes

83

8/12/2019 Outline Ch 08

20/21

A. Vesicles from ER fuse with ERGIC or cisGolgi network & not with a trans cisternaB. Selective fusion occurs & is one factor that helps ensure a highly directed flow through the membranous

compartments of the cell

II. The way in which cells target vesicles to specific compartments is not fully understood, but vesicles arethought to have specificproteins in their membranes governing their movements& fusion potential

III. Summary of the steps between vesicle budding & vesicle fusion is needed to understand the nature of theproteins in vesicle membranes controlling vesicle movement & fusion

Tar&etin& .esicles to a (articular Compartment# Summary of Steps $etween.esicle $uddin& and Fusion

I. Movement of vesicle toward the specific target compartmentA. Vesicles must sometimes move large distances through cytoplasm before reaching its eventual target; these

types of movement probably are mediated largely by microtubulesB. Microtubules act like railroad tracks carrying cargo containers along a defined pathway to a predetermined

destination

II. Tethering vesicles to the target compartment the initial contacts between a transport vesicle & its targetmembrane, such as a Golgi cisterna, are thought to be mediated by so-called "tethering proteins"A. Two groups of tethering proteins have been described:

1. Rod-shaped, fibrous proteins that are capable of forming a molecular bridge between the 2 membranesover a considerable distance (50 200 nm) &

2. Large, multiprotein complexes that appear to hold the two membranes in closer proximityB. Tethering may be an early stage in process of vesicle fusion that requires specificity between vesicle &

target compartmentC. Much of this specificity may be conferred by a family of small G proteins called Rabs, which cycle betwee

an active GTP-bound state & an inactive GDP-bound state1. GTP-bound Rabs associate with membranes by a lipid anchor2. With >60 different Rab genes identified in humans, these proteins constitute the most diverse group of

proteins involved in membrane trafficking3. More importantly, different Rabs become associated with different membrane compartments4. This preferential localization gives each compartment a unique surface identity, which is required to

recruit the proteins involved in targeting specificity5. In their GTP-bound state, Rabs play a key role in vesicle targeting by recruiting specific cytosolic

tethering proteins to specific membrane surfaces6. Rabs also play a key role in regulating activities of numerous proteins involved in other aspects of

membrane trafficking,includingmotorproteins that movemembranousvesicles through cytoplasm

III. Docking vesicles to the target compartment at some point during the process leading to vesicle fusion,membranes of vesicle & target compartment become tightly apposed to one anotherA. This is result of interaction between the cytosolic regions of integral proteins of the 2 membranes

1. The key proteins that engage in these interactions are called SNAREs & they constitute a family ofmembrane proteins whose members are localized to specific subcellular compartments2. SNAREsvarya lot instructure&size, butall of themcontaina segment in their cytosolicdomain (a

SNARE motif) consisting of 60 70 amino acidsthat form a complex with another SNARE motifB. SNAREs are divided functionally into 2 categories: v-SNAREs (incorporated into transport vesicle

membranes during budding) & t-SNAREs (located in target compartment membranes)C. The best-studied SNAREs are those that mediate docking of synaptic vesicles with the presynaptic

membrane during the regulated release of neurotransmitters

84

8/12/2019 Outline Ch 08

21/21

1. Presynaptic nerve cell membrane contains 2 t-SNAREs: syntaxin & SNAP-25, while the synapticvesicle membrane contains a single v-SNARE, synaptobrevin

2. As synaptic vesicle & presynaptic membranes approach one another, the SNARE motifs of t- & v-SNARE moleculesfromapposing membranesinteract with one anotherto form 4-stranded bundles

3. Each bundle consists of 2 -helices donated by SNAP-25 & 1 -helix donated by both syntaxin &synaptobrevin

4. These parallel -helices zip together to form a tightly interwoven complex that pulls the two apposinglipid bilayers into very close association

5. The formation of similar 4-stranded helical bundles occurs among other SNAREs at other sitesthroughout the cell, wherever membranes are destined to fuse

D. Interestingly, the SNAREs of synaptic vesicle & presynaptic membranes are targets of two of the mostpotent bacterial toxins, those responsible for botulism & tetanus1. These deadly toxins act as proteases, whose only known substrates are SNAREs2. Cleavage of the neuronal SNAREs blocks the release of neurotransmitters, which causes paralysis

IV. Fusion between vesicle & target membranesA. When artificial lipid vesicles (liposomes) containing purified t-SNAREs are mixed with liposomes

containing a purified v-SNARE, the two types of vesicles fuse with one another but not themselves

1. This finding indicates that interactions between v- & t-SNAREs are capable of pulling two lipid bilayertogether with sufficient force to cause them to fuse2. Evidence suggests that while an interaction between v- & t-SNAREs is required for fusion, it is not

sufficient alone to bring about fusion within a cellB. The prevailing view regarding the regulated secretion of neurotransmitter molecules

1. The 4-stranded SNARE bundle remains locked in an inactive conformation by interaction withaccessory proteins

2. Vesicles at this stage remain docked at the membrane & ready to discharge their contents almostinstantaneously once they receive an activating signal in the form of a rise in Ca 2+concentration

3. Regardless of how it is regulated, once membrane fusion occurs, the SNAREs that previously projectedfrom separate membranes become situated in the same membrane

4. Dissociation of 4-stranded SNARE complex is achieved by doughnut-shaped, cytosolic protein calledNSF that attaches to the SNARE bundle &, using energy from ATP hydrolysis, twists it apart

C. How is specificity of this interaction determined? current consensus is that the ability of a particularvesicle & target membrane to fuse is determined by the specific combination of interacting proteins1. The proteins include tethering proteins, Rabs & SNAREs; that can be assembled at that site in cell2. Taken together, these multiple interactions between several types of proteins provide a high level of

specificity