Endoplasmic Reticulum: Role in biosynthesis of membrane lipids, proteins and glycoproteins

Endoplasmic reticulumThe word is descriptive: Endoplasmic within the cytoplasmReticulum networkER consists of interconnecting membranous network of vesicles, tubules and flattened sacs called cisternae

Two types of endoplasmic reticulum The ER membrane is thinner than the plasma membrane and is about 5-6nm thickThere are two ERs distinguishable by prescence or absence of membrane bound ribosomesRough ER: network of vesicles, tubules and flattened sacs with ribosomes on the surfaceSmooth ER: tubular in shape without ribosomes on the surface

Rough and Smooth ER Rough Endoplasmic Reticulum (RER)

Proteins made on RER ribosomes are segregated away from the cytoplasm and can be chemically modified

Smooth Endoplasmic Reticulum (SER) - Smooth appearance is due to the absence of ribosomes, no protein synthesis - Chemically modifies small molecules taken into the cell - Site of glycogen hydrolysis and steroid synthesis

Membranes of the ER are continuous with the nuclear membrane

Rough and Smooth ER

0.5 micrometerssmooth endoplasmic reticulumvesiclesribosomesrough endoplasmic reticulum0.5 micrometers

0.5 micrometersribosomesrough endoplasmic reticulum

0.5 micrometerssmooth endoplasmic reticulumvesicles

Functions of ERThe endoplasmic reticulum serves many general functions, including the facilitation of protein folding and the transport of synthesized proteins Rough Endoplasmic Reticulummanufactures membranous and secretory proteinsThe rough and smooth ER are usually interconnected and the proteins and membranes made by the rough ER move into the smooth ER to be transferred to other locations.

Functions of the ERStarting point for newly synthesized proteins destined for Golgi, Endosome, Lysosomes, Secretory vesicles, and the Plasma membrane (see below). Establishes orientation of proteins in the membrane.Site of phospholipid and cholesterol synthesis.Initiation site for N-linked glycosylation of proteins.Sequesters Ca++ - sarcoplasmic reticulum in muscle is a specialized ER.

Smooth Endoplasmic Reticulum

SER has a wide range of functions including carbohydrate and lipid synthesis. It serves as a transitional area for vesicles that transport ER products to various destinations. In liver cells the smooth ER produces enzymes that help to detoxify certain compounds. In muscles, the smooth ER assists in the contraction of muscle cells, and in brain cells it synthesizes male and female hormones.

Sarcoplasmic Reticulumis a special type of smooth ER found in smooth and striated muscleThe only structural difference between this organelle and the smooth endoplasmic reticulum is the different proteins they have, both bound to their membranesThis fundamental difference is indicative of their functions: The smooth endoplasmic reticulum synthesizes molecules, while the sarcoplasmic reticulum stores and pumps calcium ions

SYNTHESIS OF PHOSPHOLIPIDS

PHOSPHATIDYL-CHOLINE: synthesisCHOLINE:Food sourcesCirculationPresynaptic cholinergic membraneCholine-transport - high affinity Synthesis of phosphatidyl-choline in hepatocytes from phosphatidyl-ethanolamine*

The synthesis of phosphatidylcholine

MOVEMENT OF PHOSPHOLIPIDS FROM ER TO OTHER ORGANELLESSynthesized lipids are moved to plasma membrane via Golgi by vesicular transportThis mode of transport involves vesicles containing phospholipids, proteins etc budding off the ER, fuse with the Golgi and then budding off the Golgi and then fusing with the target (plasma) membraneMovement of phospholipid from membrane of one organelle (eg ER) to mitochondria is by phospholipid exchange proteins. These remove phospholipid from cytosolic leaflet of the ER to the cytosolic leaflet of target membrane

Transfer of lipids to other organelles.Most lipids for other organelles are synthesized at the ER.Lateral diffusion will supply the nuclear membrane.Vesicular transport will supply organelles in the secretory pathway and lysosomes (vesicular transport will be described soon)Phospholipid exchange proteins deliver phospholipids to the mitochondria, chloroplasts and peroxisomes.

Role of ER in protein synthesis and glycosylation

ER role in protein synthesis and targeting Two types of ribosomes exist in the cellFree ribosomes in the cytosol (often bound to cytoskeletal fibres): These synthesize soluble cytosolic proteins such as glycolytic enzymes and cytoskeletal proteins Membrane bound ribosomes attached to the RER which synthesize three major classes of proteinsSecretory proteins proteins exported outside the cellProteins localized to the ER, Golgi, and the plasma membraneIntegral membrane proteins of the plasma membrane (integral membrane proteins except those of the mitochondria and chloroplasts are made by ER bound ribosomes) Note: there is no structural difference between cytosol free ribosomes and ER bound ribosomes. The differences exists only in the particular protein being synthesized at a particular timeER bound ribosomes synthesizes proteins with signal sequences

Signal sequencesThe attachment of an actively synthesizing ribosome to the ER is a key event in translocation of the protein across the ER membrane to the ER lumenThe signal for attachment is a sequence of amino acid residues at the N-terminus of the growing (nascent) polypeptide called a signal sequenceNote: signal sequences are absent from secreted proteins because they are cleaved by a signal peptidase on the luminal side of the ER membrane

Signal sequencesSignal sequences have several common featuresThey range in length from 13-36 amino acidsThe amino-terminal part of the signal sequence contains at least one positively charged residue A highly hydrophobic stretch of 10-15 residues long form the center of the signal sequence. Alanine, leucine, valine, isoleucine and phenylalanine are common in this region. Substitution of any hydrophobic residue with a charged residue destroys the directing activity of the signal sequenceThe residue on the amino terminal side of the cleavage site usually has a small neutral side chain (alanine is common)

Secreted Proteins Have N-Terminal Signal SequenceSignal sequence is 10-15 residue hydrophobic stretch near N-terminus.

Signal sequence triggers secretion mechanism, is usually cleaved at nearby downstream small amino acid (Gly-X, Ser-X, Ala-X)

Signal sequences that target proteins to different locations in bacteria+ chargehydrophobic

Signal recognition particleSignal recognition particle (SRP) detects signal sequences and brings ribosomes to the ER membrane ie SRP binds tightly to ribosomes containing a nascent pptide chain with a signal sequence but not to other ribosomesSRP is a 325 Kda assembly consisting of a 300 nucleotides RNA 7SLRNA and six polypeptide chains: P9, P14, P19, P54, P68, P72P54 binds to signal sequnces and is the only one that does not associate with the 7SLRNAP68 and P72 are required for protein translocation in the lumen of ERP9 and P14 interacts with the ribosomes

Methionine "whiskers" on P54 subunit bind to the hydrophobic signal sequence on the emerging polypeptide

SRP is a ribonucleoprotein300 base RNA molecule6 proteins

SRP receptorThe complex consisting of SRP, nascent polypeptide and the ribosome binds to SRP receptor in the ER membraneSRP receptor is an integral membrane protein of two subunits, subunit (68 KDa) and subunit (30 KDa)Binding of SRP-signal peptide to receptor triggers GTP exchange for GDP bound to the subunit Once the GTP form of the receptor binds SRP, this releases its grip on the signal peptide which swiftly binds to the translocation machinery (translocon)Hydrolysis of GTP to GDP by subunit release the SRP which can resume another cycle

SRP receptor initiates the interaction of signal sequences with the ER membraneReceptor is an a,b dimer b subunit is an intrinsic membrane protein a-subunit initiates binding of ribosome SRP to ER membrane

GTP hydrolysis powers 1) dissociation of SRP, SRP receptor from translocon, 2) opening of translocon gate, 3) transfer of signal sequence to translocon

Steps in translocation of nascent protein across ER membraneSteps 1 and 2: Signal sequence emerge from ribosome bound to SRPStep 3: SRP delivers the ribosome plus nascent polypeptide to SRP receptorStep 4: transfer of ribosome plus nascent pptide to translocon. SRP docks to and opens the translocon. SRP and SRP receptor dissociate from translocon. The bound GTP on subunit is hydrolysed. The released SRP can initiate insertion of another pptide chainStep 5: pptide elongates through translocon channel and the signal sequence is cleaved by a signal peptidaseStep 6: Elongation continues as mRNA is translated towards 3 end. The growing chain is extruded through transloconSteps 7 and 8: translation completed, ribosome released, translocon closes, protein folded into its native conformation

Post-translational modifications and quality control in the rough ERNewly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications1. Formation of disulfide bonds2. Proper folding3. Addition and processing of carbohydrates4. Specific proteolytic cleavages5. Assembly into multimeric proteinsModifications 1, 2 and 5 takes place exclusively in the ERModifications 3 and 4: some processes occur in the ER, the rest in the Golgi or in the organelle the protein is taken to

Disulfide bond formationDisulfide bonds (Cys-S-S-Cys) always occur in the lumen of ER. Never in the cytosolDisulfide bonds are important as stabilizing forces in tertially structures of secreted and some membrane proteinsGlutathione, the major thiol (SH) containing molecule serve 2 purposes:Prevent formation of disulfide bonds in the cytosolCatalyse formation of disulfide bonds in the lumen of ER, the bonds form by a thiol-dependent exchange mechanism with oxidized glutathione

Disulfide bond formationCH2-SHCH2-SH+ G-S-S-GG-SHCH2-S-S-GCH2-SHG-SHCH2-SCH2-SIn ER lumen the ratio of G-SH to G-S-S-G is maintained at 5:1 while that of the cytosol is 50:1. This is optimal for disulfide bond formation in the lumen and not cytosol

Once the protein has been synthesized there is rearagementof these bonds by a protein called protein disulfide isomerase.The role of this protein is to catalyse rearagement of these bondsuntil a thermodynamically most stable configuration is reached

Rearagement of disulfide bondsProtein disulfide isomerase contain an active site cysteine residue with a free sulfhydryl group (SH)This can react with disulfide (S-S) bonds in nascent and newly synthesized proteins to form new S-S bond between PDI and the proteinThe bond, can in turn, react with free SH in the protein to create a new bondThis rearagement continue until the most stable configuration in the protein is reached

Disulfide bonds are formed and rearranged in the ER lumen

Protein foldingSecretory proteins generally fold in the ERER contains several proteins that facilitate foldingChaperonesLectins (calnexin)Peptidylprolyl isomerases

Protein foldingThe ER lumen contains resident chaperons that bind nascent proteins and assist in their folding. This prevents the nascent proteins becoming entangled with each other because protein concentration is very high (200mg/ml)One of the resident chaperons is a protein called BIP (binding protein), a 78KDa protein and a member of the heat shock proteinsBIP is an ATPase with two domains, an ATP binding and a peptide binding domainWhen bound to ADP, the chaperon has a high affinity to unfolded proteins but not native ones. This stimulates release of ADP and entry of ATP to the chaperon catalytic siteThe hydrolysis of ATP enable the chaperon to bind again to another unfolded segment of the protein. This is repeated until the protein is completely folded

Folding quality controlOnly properly folded proteins can be transported from rough ER to GolgiMisfolded or unassembled proteins are retained in the ER bound to chaperones or lectinsThey are degraded or transported back to cytosol for degradation

Formation of multimeric proteinsMany secretory and membrane proteins are oligomers (ie built of two or more polypeptides)Occurs prior to export to GolgiOligomerization occurs by self-assemblyInvolves chaperones that protect hydrophilic surfaces until contact is possible (subunits may be products of different genes and maybe translated with different speed)

GlycosylationMajor biosynthetic function of ER- proteins transported into other intracellular locations eg Golgi, lysozomes and plasma membrane are already glycosylatedGlycosylation is a reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) It is an enzymatic process that attaches glycans to proteins, lipids, or other organic moleculesIt is a form of co-translational and post-translational modification of proteinsThere are two major types of glycosylation: N-linked and O-linked glycosylation

N-linked glycosylation - Most common type of glycosylation present in glycoproteins - Sugar attached to Asparagine Residue. - Consensus peptide sequence is Asn X Ser or Thr - Common Sugars attached are N- Acetylglucosamine (GlcNAc), and Mannose - Glycosylation occurs cotranslationally, in the Rough ER.

O-linked glycosylation

- Sugars attached to Serine/Threonine residues. - Common sugars attached are N-Acetyl Neuraminic (Sialic) Acid and N-Acetyl galactoamine - Glycosylation occurs posttranslationally, in the Golgi.

Proteins are Glycosylated in ER and Golgi-important for folding

-important for function

-important for targeting

Purpose of glycosylationThe carbohydrate chains attached to the target proteins serve various functions:Folding: some proteins do not fold correctly unless they are glycosylated first.Stability: confer stability on some secreted glycoproteinsProtein degradation: unglycosylated protein degrades quicklyCell-cell interaction: Glycosylation plays a role in cell-cell adhesion (a mechanism employed by cells of the immune system) via sugar-binding proteins called lectins, which recognize specific carbohydrate moieties

N-linked glycosylationTransfer of oligosaccharide to asparagine occur on the lumenal side of the ER membrane by a reaction catalysed by a membrane bound transferaseThe oligosaccharide is preformed first in the cytosol and finally in the ER lumen and then transferred to target asparagine in a single stepThe target asparagine are those with Asn-X-Ser or Asn-X-Thr, where X is any amino acidThe oligosaccharide to be transferred is present in an activated form by linking it to a donor molecule via a high energy bondThe activated donor molecule is a lipid called dolichol phosphate to which the oligosaccharide is linked via a pyrophosphate bridge

Dolichol-P and N-linked glycosylation

The core oligosaccharide used for N-linked glycosylation is assembled onto the polyisoprenoid lipid, dolichol pyrophosphateIn vertebrate tissues, dolichol contains 18-20 isoprenoid units (90-100 carbons total).

Dolichol phosphate

Formation of the Core Oligosaccharide on Dolichol Phosphate starts in the cytosol and is completed in the ER lumenAll the sugar groups attached to asparagines have a common core structure That consists of 3 glucose, Nine mannose and 2 N-acetyl glucosamine units. The sugar linked directly to asparagine is N-acetyl glucosamine

Blocked by Tunicamycin, a Hydrophobic analog ofUDP- N-acetyl glucosamineTunicamycin is a Streptomyces compound that blocks transfer of GlcNAc-1-P from UDP-GlcNAc to dolichyl-PFormation of the Core OligosaccharideThe dolichol pyrophosphate released isconvert to dolicol phosphate by aphosphatase and recycled. This hydrolysisis blocked by the antibiotic bacitracin

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Tunicamycin mimics UDP-acetylglucosamine and block the first of oligo synthesis

Synthesis of core oligosaccharide and transfer to nascent polypeptide

Quality control in the ERGlucosylation prevents unfolded or misfolded proteins to be retained in the ERAfter the glycoprotein is synthesized, two glucose residues are removed while the protein is in the ER by glucosidase I and IIThis glucosylated glycoprotein binds to the chaperon calnexin which together with ERp57 assist in proper folding. Calnexin prevent secretion of the protein until it is properly folded. If the protein is properly folded the third glucose is removed and the protein exportedIf the N-linked glycoprotein is unfolded or misfolded, one glucose residue is added back by a glucosyl transferase. The protein bind calnexin which prevent its export and will assist with its foldingCalnexin thus prevents export of immature and defective proteins and this kind of quality control system ensures that the glycoprotein is ready for export to the Golgi complex

Quality Control in the ER Calnexin/calreticulin bind to incompletely folded monoglucosylated glycans

Cycles of binding/release controlled by: Glucosidase II: cleaves glucose from core glycan

UDP-glucose: glucosyltransferase (GT) reglucosylates incompletely-folded proteins so that they bind lectins again

Thus GT acts as a folding sensor: proteins exit the cycle when GT fails to re-glucosylate. Glucose is a tag that signifies incomplete folding

10-9Folding in the Endoplasmic reticulum

Retention of ER proteinsER resident proteins such as BIP chaperon and protein dislufide isomerase are retained in the ER and not secreted with other proteinsThese proteins contain a specific sequence lys-asp-glu-leu (KDEL) at the C-terminus The KDEL sequence binds to a receptor protein and the protein is retained in ERProteins with KDEL sequence that escape retention in ER are retrieved in the cis Golgi and brought back in vesicles containing the receptor

Retrieving ER proteins

*Figure: 04-07

Title:Endoplasmic reticulum.

Caption:There are two types of endoplasmic reticulum: rough ER, coated with ribosomes, and smooth ER, without ribosomes. Although in electron micrographs the ER looks like a series of tubes and sacs, it is actually a maze of folded sheets and interlocking channels.

*Figure: 04-07R-1

Title:Rough endoplasmic reticulum.

Caption:Rough endoplasmic reticulum.

*Figure: 04-07R-2

Title:Smooth endoplasmic reticulum.

Caption:Smooth endoplasmic reticulum.

*****Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. SRP shows 3 main activities in the process of cotranslational targeting: first, it binds to signal sequences emerging from the translating ribosome; second, it pauses peptide elongation; and third, it promotes protein translocation by docking to the membrane-bound SRP receptor and transferring the ribosome nascent chain complex (RNC) to the protein-conducting channel.

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