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Unformatted text preview: The secretory pathway and the biogenesis of intracellular membranes- • Phospholipid synthesis in the ER • Protein insertion into the ER • Protein glycosylation • Protein folding in the ER • Quality control in the ER • Protein sorting in the smooth ER and Golgi • Vesicle-mediated transport ER -> Golgi -> PM • Regulated membrane fusion • Directionality in vesicle trafficking The endoplasmic reticulum is the entry-point for proteins into the secretory pathway Vesicles mediate protein transport between membranes in cells- Clathrin COPI vesicle coats COPII Exomer Elements of specificity in the surface of transport vesicle and organelle membranes• • • • Rab proteins Tetherins/TRAPPs vSNAREs tSNAREs Rabs are membrane-specific markers and each also has its entourage of membrane specific GAPs and GEFs A different Rab GTPase protein marks each compartment of the secretory and endocytic pathways Tetherins, vSNAREs, and tSNAREs are also localized to distinct compartments How to study the molecular machinery underlying the secretory pathway? • Morphological examination of cells How to visually follow a secretory protein? • Isolation of mutants What phenotypes would you expect? • In vitro reconstitution How to track biochemically the movement of secretory proteins between compartments? A morphological approachFirst demonstration that intracellular membrane compartments form part of the secretory pathway… Secretion Nobel Prize 1974 to George Palade Palade was interested in ribosomes, and wondered what the relationship was between them and intracellular membranes Classic experiment: Autoradiography of pancreatic acinar cells- Radiolabeled proteins were observed as autoradiographic grains over the ER at the time of pulse (time 0), over the Golgi compartment after 7 min of chase, and over secretory vesicles after 80 min. 0 min ER 7 min Golgi 80 min Secretory vesicles The ER is a vast and dynamic organelle where secretory proteins are modified with carbohydrates that facilitate their folding
The rough ER is coated with ribosomes translating proteins destined for secretion Carbohydrate synthesis, addition, and trimming in the endoplasmic reticulumWhat are the carbohydrates good for? • Protein folding and quality-control • To protect the protein (by coating it) • Involved in cell identity and recognition Biosynthesis of the N-linked oligosaccharide in the ER. 1. 2. 3. 4. 5. Starts with the sequential addition of seven monosaccharides (GlcNAc-phosphate, GlcNAc, and five mannose residues) to a dolicholmonophosphate on the cytosolic surface of the ER membrane. This lipid-linked oligosaccharide is flipped into the lumenal side of the membrane, and four mannose and three glucose residues are then added. The core oligosaccharide is then transferred to the asparagines residues of nascent growing polypeptides mediated by the oligosaccharyl-transferase complex. The three glucose residues are trimmed away by glucosidase I and II (GI and GII) (quality control) After the protein has folded and assembled in the ER, the protein is moved to the Golgi complex and the N-glycans receive further modification. The Golgi membranes are specialized compartments for protein glycosylation
Morphology of Golgi membranes and its surrounding vesicles Carbohydrate modification of proteins along the secretory pathway (a useful diagnostic tool for tracking the movement of proteins along the path) Sugar precursors are imported into the ER and Golgi lumen by antiporters Example of precursors are: UDP-mannose, UDP-GlcNac, CMP-sialic acid Useful enzymes in biochemical analysis of glycosylated proteins; useful for diagnosis of passage through the secretory pathwayetc etc GlcNAc GlcNAc
endoH N-glycanse GlcNAc GlcNAc
N-Glycanase NH CH 2 C O CH 2 C O C O N H C O N H peptide-Asn peptide-Asp Endo H cleaves between the N-acetyl glucosamines of core carbohydrates. Upon arrival to the cis-Golgi the carbohydrate chain is modified in a way that becomes resistant to Endo H action. Thus, Endo H resistance is used as a marker for arrival of a N-linked glycosylated secretory protein to the cis-Golgi compartment. N-glycanase cleaves the entire N-linked carbohydrate converting asparagine into aspartic acid. Cleaves regardless of the modifications to the carbohydrate. Used to determine if a protein is N-linked glycosylated or not. Neuraminidase cleaves the sialic acids from the terminal branches, which are added in the trans-Golgi compartment; thus it is used as a diagnostic tool for “arrival to the trans-Golgi. Genetic approach to isolate mutant cells blocked in protein translocation across the ER membrane- Genes identified:
Sec61 Sec62 Components of ER translocon Sec63 Sec65 - component of SRP
…a histidine auxotroph Signal sequence A mutation in a translocon subunit could slow down, but not prevent translocation A genetic approach to dissect the secretory pathwayScreen for temperature-sensitive conditional mutants in yeast
1) S cerevisiae cells (yeast) are forced to mutate by short exposure to a mutagen (UV light, EMS, etc.). 2) Mutant cells that are partially blocked in secretion become dense because their protein-to-lipid ratio is altered. Mutant cells were selected among the most dense cells in density gradients. 3) Only mutants with temperature sensitive (30°C vs 37°C) defects were chosen, since secretion is essential for survival of yeast. Thermoreversible. 4) Protein secretion was blocked in the mutants; diagnosed by intracellular accumulation of secreted proteins. 5) Morphological examination of sec mutants at the non-permissive temperature revealed an accumulation of intermediate organelles of the secretory pathway. 6) Sec gene products (Sec proteins) were identified by reversal of the phenotypes using genetic complementation with plasmid libraries Distinct classes of mutants in the secretory pathway- Enlarged ER Enlarged Golgi Sec mutant combinations and morphological examination were used to identify sequential intermediates in the secretory pathwayA morphological ‘ordering’ experiment: enlarged ER enlarged Golgi enlarged ER Secreted proteins are held within the cells, and display only ER-specific core carbohydrate structures Secreted proteins are held within the cells, and display only Golgi-specific carbohydrate structures Secreted proteins are held within the cells, and display only ER-specific core carbohydrate structures Conclusions: A biochemical assessment of secreted protein location Intermediate A occurs before intermediate B The function of gene product A is needed before the function of gene product B A transient ‘vesicle intermediate’ in the ER-to-Golgi secretory pathway- Biochemical approach: Reconstitution of ER to Golgi transport- In vitro translation of pre-pro-alpha -factor Post translational translocation at low temperature: 10°C ER modified formRecognized by lectin Concanavalin A (ConA) Transport at 25°C in the presence of cytosol and ATP /GTP regeneration system Golgi modified formRecognized by anti-alpha 1,6 mannose linkage specific antibodies, and by ConA Both forms are ‘protease protected’ ER to Golgi transport25% efficiency Percent efficiency of transport = (Golgi form/ER form) x 100 Reconstituted ER to Golgi transport requires cytosolic Sec proteins- Biochemical reconstitution of protein transport between Golgi stacks- Follow glycosylation state of VSV -G protein (a viral protein) as evidence of transit between cis and medial Golgi membranes Assumes VSV-G is transported in the anterograde direction between Golgi stacks via vesicles This assay was used to identify and purify NSF, SNAPS, and SNAREs involved in vesicle membrane fusion with the target organelle Vesicular transport between Golgi stacks versus cisternal maturationThe case of Pro-collagen and fish scales… secreted proteins that are too big to fit in vesicles Consider the interpretation of the cis to medial Golgi assay assuming that transport through the Golgi proceeds via cisternal maturation… ...
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This note was uploaded on 01/17/2011 for the course BIOL 110 taught by Professor Rexach during the Fall '10 term at UCSC.
- Fall '10