Lecture 8 - Exam Flashcards
What techniques were/are used to determine which proteins moved from organelle to organelle in the secretory pathway?
Pulse-chase labeling experiments, autoradiography, fluorescent microscopy of cells with GFP membrane tag (modern)
Are proteins released into the cytosol?
No, they are always associated with membrane-bound intermediates
How long does mammalian cell vesicle-mediated transport take from ER to PM?
30-60 min
What is VSVG and what is it used for?
Vesicular stomatitis virus glycoprotein labeled with GFP is used to monitor protein transport in the secretory pathway
What is restrictive temperature VSVG?
A type of mutant at 40 degree C VSVG-GFP is retained in the ER
What is permissive temperature VSVG?
A type of mutant at 32 degree C VSVG-GFP is released for transport to golgi and plasma membrane
How can transport of a membrane glycoprotein from ER to golgi be assayed?
Sensitivity to cleavage by endoglycosidase D - can be run on SDS-PAGE
What do many secretory proteins leaving the ER have as side chains? And what cleaves them and what does it produce?
One or more copies of N-linked oligosaccharides (carbohydrates) -
Mannose N-acetyl glucosamine [Man8(GlcNAc)2] - cleaved by glycosidases on golgi cisternae that trim to [Man5(GlcNAc)2]
What identified the five stages in the secretory pathway?
Phenotypes of yeast sec mutants - organization and components is similar in all euk - temperature sensitive sec mutants are defective for secretion of proteins at non-permissive termperatures - 5 classes A-E showed sites where newly synthesized proteins accumulate when sec mutants are moved from permissive to non-permissive temperatures
What are the 5 stages in the secretory pathway?
Cytosol –> rough ER –> ER-Golgi transport vesicles –> golgi –> secretory vesicles
What demonstrates protein transport from one golgi cisternae to another? And how does it happen?
Cell-free assay - isolated golgi from VSV wildtype and mutant cells - WT contains N-acetylglucosamine transferase I enzyme that transfers to a mannose residue and mutant does not, if you mix the two extracts together the WT enzyme is still capable of transfering this n-acetyle glucosamine to the mutant by moving across cisternae
What initiates budding in the secretory pathway?
GTP-binding proteins
What gives the curvature of a vesicle bud in the secretory pathway?
Coat proteins - drive formation of structure
What interaction allows for secretory pathway vesicle bud fusion?
v-SNARE proteins on donor membrane that undergo a conformational change to interact with t-SNARE proteins
What do membrane cargo proteins do in secretory vesicles?
They have cytosolic sequences that determine what kind of coat should be made
What do membrane cargo-receptor proteins do in secretory vesicles?
Interact with soluble cargo proteins needed for vesicle formation
What is the most well known and widely distributed coat protein for vesicles
Clathrin and AP adapter protein complexes
What is the main GTPase associated with budding?
ARF
What are the different vesicle types and associated GTPase and coat proteins ?
COPII - Sar1, uses coat proteins sec
COPI - ARF, uses coatomers as coat proteins
Clathrin - ARF, uses APs and clathrin as coat proteins
Explain assembly and disassembly of COPII coats
Sec12 (GEF for Sar1) causes GDP-GTP exchange undergoing conformational change to bind to ER membrane, Sar1-GTP polymerizes Sec23/Sec24 COPII subunits to lead to vesicle budding, COPII vesicles are released, GTP hydrolyzed to GDP, COPII coat dissassembles
What prevents coats from not disassembling?
If GTP can not be hydrolyzed - using something like GTP-gamma-S - causes accumulation of coated vesicles
How do vesicles know where to go?
Cargo protein targeting sequences make specific contacts with coat proteins
Luminal sorting signals - necessary sorting signal since soluble proteins can not contact coat directly (KDEL and M6P are examples)
Cytoplasmic sorting signmals - are specific sequences in cytosolic portion of membrane cargo proteins that interact with coat (*KKXX, NPXY, LL are examples)
What is a model pathway for docking and fusion of transport vesicles?
Rab-GTP protein bound to secretory vesicle by lipid anchor binds effector protein on PM allowing docking –> v-SNARE (VAMP) interacts with t-SNARE (syntaxin, SNAP-25) to form stable complex to hold vesicle close to target membrane –> two membranes undergo fusion –> NSF and alpha-SNAP bind to SNARE complex and NSF (ATPase) hydrolyzes ATP to ADSP and drives dissociation of SNARE comples, Rab-GTP is hydrolyzed and dissociates from Rab effector
What type of vesicles mediate anterograde transport from ER to golgi?
COPII
What type of vesicles mediate retrograde transport from golgi to ER?
COPI
What type of vesicles mediate transport to endosomes, melanosomes, lysosomes or platelet vesicles?
Clatherins
How can ER proteins that mistakenly go to the golgi be retrieved?
Proteins have KDEL sorting sequence that binds KDEL receptor in golgi in COPII and COPI vesicles and returns them to the ER - pH sensitive where golgi has a lower pH giving higher binding affinity and ER has higher pH which induces peptide release
What processing of N-linked oligosaccharide chains on glycoproteins within cis cisternae of goli occurs?
Removal of 3 mannose residues
What processing of N-linked oligosaccharide chains on glycoproteins within medial cisternae of goli occurs?
N-acetylglucosamine residue is added to 5 mannose (from cis)
2 more mannose are removed
Two more N-acettylgucosamine residues added
Fucose residue added
What processing of N-linked oligosaccharide chains on glycoproteins within trans cisternae of goli occurs?
Three galactose residue added
Linkage of N-acetylneuraminic acid residue to each galactose residue
What types of enzyme are involved in modifying the N-linked carbohydrates attached to secretory proteins?
Glycosidases and glycosyltransferases
What is the process of membrane proteins advancing through the golgi complex called? and how can it be visualized?
Cisternal maturation by anterograde transport - visualized by fluorescent tagging in living yeast cells
Where can proteins go after being in the trans-golgi network?
Retrograde transport via COPI vesicles if mistakenly moved
Moved to endosomes and lysosomes by clathrin and AP vesicles
Cell surface - coat not characterized
What is the structure of clathrin coats?
Triskelion structure with 3 heavy chains and light chains with two layers Adapter protein (AP) complexes and clathrin molecules (form the triskelion)
What is the name of the GTPase responsible for pinching off clathrin/AP coated vesicles?
Dynamin
What targets the transport of lysosomal enzymes in vesicles to endosomes? and where is it generated?
Mannose 6-phosphate (M6P) - a sorting signal that directs lysosomal enzymes for trans-golgi to late endosomes when they are attempted to be brought to PM - generated in cis-golgi but receptor sits on PM
Where can lysosomal enzymes by trafficked?
Can be secreted to cell surface but recognized by M6P to be brough to endosome and be brough back to lysosome
Can be brought right to endosomes to be transported to lysosome
What is the only apical sorting signal identified so far?
GPI-anchor
How do proteins know whether to go to the apical or basolateral side and what are some examples of proteins that have been tested in experiments?
Sorting signals - basolateral sorting signals have been identified much easier - VSVG glycoprotein always goes to basolateral, but influenza virus always goes to apical side
What is the process called when proteins are moved from basolateral to apical sides?
Transcytosis - proteins are endocytosed and moved laterally across cell
Where do all PM proteins go?
Basolateral
What is the most well document receptor-mediated endocytosis?
Low-density lipoprotein (LDL) -
What is receptor-mediated endocytosis (RME)?
When a specific receptor on the cell surface binds tightly to an extracellular macromolecular ligand that reconizes it –> PM regions containing receptor complex buds inward and pinches off becoming transport vesicle –> occurs via clathrin/AP2 vesicles
What is added to visualize transport/vesicles in electron micrographs?
Gold or ferritin
What is the structure of LDL?
Amphipathic structure with hydrophilic outer surface and hydrophobic inner surface, apolar core made up of cholesterol with polar surface made up of phopholipids and wrapped by apolipoprotein B (ApoB)
What is the structure of the LDL receptor?
It is a glycoprotein with transmembrane segment, with the exoplasmic domain containing the LDL-binding domain (7 Cys repeats) - cytosolic domain contains LDLR sorting signal to incorporate receptor-ligand complex into endocytic vesicles
What is the general endocytic pathway for internalizing LDL?
LDL particle binds LDL receptor at ApoB with Cys 4 and 5 in ligand binding arm and at pH 7 and vesicle envaginates –> Clathrin/AP2 vesicle uncoats forming early endosome with low pH 5 causing dissociation of receptor-ligand complexes when His residue in beta propeller domain becomes positively charged and binds the ligand binding arm –> late endosome recycles LDL receptor and delivers cholesterol, AA and FAs to lysosome
What is transferrin? and what is it’s cycle
A major glycoprotein in blood that transports iron to all tissue cells - apotransferrin (iron free form) binds two iron ions tights to form ferrotransferrin –> transferrin receptor binds ferrotransferrin at neutral pH –> complex is endocytosed and in the late endosome at pH 5 iron ions dissociate and arm reduced to Fe2+ from Fe3+ –> the apotransferrin returns to the cell surface and is secrets where it dissociates from receptor at neutral pH
How are PM proteins delived to lysosomal interior?
Endocytosed proteins are carried by early endosomes carrying lysosomal enzymes from trans-golgi and fuse with late endosomes transferring cargo –> proteins to be degraded are incorporated into vesicles that bud inside late endosome forming multivesicular endosome –> this fuses with lysosome and releases internal vesicals and are degraded –> those not to be degraded are not incorporated into these vesicles and delivered to lysosomal membranes
How are multivesicular endosomes formed?
Ubiquitin tags proteins for entry –> Hrs (a Ub-tagged peripheral membrane protein) recruits endosomal sorting complexes required for transport ESCRT –> ESCRT drive vesicle budding directed into interior of endosome and load Ub-proteins into buds —> ESCTR pinches off vesicle and ATPase Vps4 disassembles complex
How does HIV bud from the PM?
HIV is an enveloped retrovirus that buds from the PM of infected cells in a process driven by Gag protein and ESCRT and Vps4 ATPase - Gag polymerize spherical shell generating structure similar to vesicle bud
