MCB 2210 001 3 6 2015 Proteins on the cytoplasmic face of vesicles regulate budding and fusion 1 Budding o The mechanical process of membrane budding is driven by a complex of cytoplasmic proteins that assembles on the outside cytoplasmic face of budding vesicles They coat the vesicle surface so are called coat proteins o Different vesicle types have different coat proteins COPII coats vesicles moving ER to Golgi retrograde transport backwards COPI coats vesicles moving Golgi to ER and from more trans Golgi to more cis Golgi anterograde direction Clathrin coats vesicles moving from TGN to surface and from surface to endosomes lysosomes Endocytosis o The contents of the vesicle apparently play no direct role in triggering budding Different Coated Vesicles can be distinguished morphologically A class of small G proteins regulates budding o These cytoplasmic G proteins associate with membranes GTP bound budding is initiated GEF GDP bound no budding or removal of coat GAP o Different types of membrane budding going to different compartments use a different G protein COPII Sar 1 COPI Clathrin Arf o These G proteins have a covalent lipid anchor that can be exposed or hidden allowing them to reversibly associate with the cytoplasmic surface of the membrane Budding 1 Sar1 membrane binding GTP Exchange 2 COPII coat assembly 3 GTP hydrolysis 4 Coat disassembly Non hydrolyzable analogs of GTP such as GTP gamma s trap vesicles in the coated form The vesicle cargo o Some soluble proteins seem to be carried along non specifically by bulk flow o Other soluble proteins seem to bind to cargo receptors which concentrate them in vesicles These transmembrane receptors interact via cytoplasmic domain with coat proteins o Membrane proteins can themselves interact with coat proteins o Implies a signal for export is needed to interact with coat o Some proteins are specifically prevented from moving out of the ER because they are incorrectly folded Chaperones hold back misfolded proteins The signals for export in cargo binding receptors or cargo proteins themselves in AA sequences that interact with coat proteins o Cargo receptors can interact with the COPII coat proteins Export signals in cargo receptors or membrane proteins themselves interact directly with the COPII coat proteins Budding Summary o Sar1 activated COPII proteins begin to bud membrane o Cargo loaded by bulk or by direct or receptor mediated interaction with coat proteins o Vesicle buds off o COPII coat Sar1 hydrolyze GTP coat disassembles o Now need to dock and fuse Yet another class of small G proteins help control docking to target membranes o Docking and fusion are regulated by the Rab family of small G proteins Different routes of vesicle trafficking appear to be controlled by different Rab family members over 60 Together with SNARES they control the specificity of vesicular traffic ER to CGN TGN to secretory endocytosis etc GTP bound state active tightly associated with membranes or vesicles mediate tethering and docking of vesicles via a variety of effector proteins in target membrane Hydrolysis to GDP allows release of inactive Rab to cytosol Transmembrane proteins called SNAREs control fusion of the vesicle to the target membrane in vesicle membrane and in the target compartment membrane o The coats disassemble after budding This means the coat does not play a role in targeting o Vesicles have transmembrane proteins called V SNARES o These are recognized by T SNARES on the Target membrane provides directionality between compartments o Bring membranes into close apposition allowing fusion o Many unique T SNARE and V SNARE proteins exist and specific pairings mediate specific membrane fusion events o So together Rabs and SNARES control the specificity and directionality of vesicular delivery and fusion Note that that this all happens on the cytoplasmic side of the membranes ER Golgi vesicle trafficking 1 Active Sar1 G protein initiates membrane budding process 2 Recruits cytoplasmic COPII coat proteins to drive budding 3 Receptor bound proteins and free proteins are recruited into the budding vesicle 4 The vesicle buds 5 The adaptor coat falls off 6 Vesicle finds and docks its target membrane via Rab G proteins Rab effectors and SNARE proteins SNARE complexes drive fusion 7 COPII vesicle transport has thus contributed to the CGN What controls exit from or return to the ER o Proteins that enter the ER are generally able to move from the ER to the Golgi o There are specific ER export signals that can interact directly or indirectly with coat proteins These proteins move on to the Golgi and beyond o Proteins that are supposed to function in the ER are recycled back to the ER Protein Disulfide Isomerase catalyzes Cys Cys disulfide bonds Chaperones that aid in folding Bip SRP receptor o An ER retrieval signal for soluble proteins is KDEL Lys Asp Glu Leu The KDEL ER retention signal is at the C terminus KDEL binds a KDEL receptor in the Golgi KDEL receptor interacts with COPI coat proteins on cytoplasmic side o There are other ER retrieval signal sequences for membrane proteins These are not always at the C terminus and interact directly with COPI o KDEL receptor cycles between ER and Golgi Differences in pH between the ER and later compartments may control binding and unbinding to receptors Golgi is more acidic lower pH than ER o At lower acidity the KDEL retrieval sequences cannot interact with the KDEL receptor ER o In Golgi since the pH is lower higher acidity the COPII on KDEL can interact with the KDEL receptor on Golgi membranes o So pH determines whether a KDEL containing protein is taken to the ER ER retrieval signals interact directly or indirectly with COPI coat proteins to return proteins to the ER KDEL o A soluble protein buds off of ER membrane with a COPII coat o ER to Golgi transport vesicle o COPII interacts with the KDEL receptor on the Golgi o The vesicle fuses with the Golgi membrane and the protein is delivered o The vesicle uptakes retrieves the KDEL bearing proteins to ER Each Golgi compartment has a unique enzyme composition How do cargo proteins get exposed to each o Vesicle Transport Model Vesicles bud and fuse to move cargo proteins and membrane forward from cis to medial to trans Golgi compartment So vesicles can be used to transport proteins within the Golgi complex itself A given compartment and its processing enzymes stay constant as cargo trafficked through o Problems Only COP I vesicles which are known