Exam 2 will be held Friday October 23rd in the Tes ng Center Arjona 110 You must sign up for an available Exam Slot on HuskyCT Exam Registra on Link There is no regular class on exam day If you have another class con ict with all the available slots you must provide a screenshot of your UConn schedule and contact me ASAP HuskyCT Materials will be unavailable online that Friday Be sure to download any materials you want if you plan on last minute studying There will be 2 Review sessions before the Exam 1 Wednesday 10 21 6 8pm TLS 154 2 Thursday 10 22 6 8pm TLS 154 Bring your Problem Sets The early exam for people with legi mate reasons that have been cleared with me will be on Thursday 10 22 Time place TBA If you need to take the early exam for a legiImate reason and have NOT contacted me yet please do so ASAP david daggeO uconn edu Students who have CSD Exam me place accommoda ons will be taking the exam at the CSD o ce Please get any Exam AdministraIon Forms to me ASAP From the ER to the Golgi Apparatus Vesicle Tra cking 1 3 2 4 Transport Mechanisms Gated Transport Transport between topologically equivalent compartments but entry and exit controlled Nucleus nuclear pore Transmembrane Transport Membrane bound protein translocators move proteins across the membrane ER mitochondria chloroplast Vesicular transport Membrane enclosed vesicles bud and fuse to move proteins from compartment to compartment ER to Golgi and beyond endocytosis Flow through the secretory pathway can be visualized with GFP GFP tagged viral protein VSV G that is expressed in membrane of host cells Temperature sensiIve mutant is blocked in transport and accumulates in ER at 40 deg C Then block is released at 32 deg C allowing protein to move to Golgi and then plasma membrane Yeast mutants have been incredibly useful in understanding the secretory pathway Four views of the Golgi apparatus Golgi Apparatus Modi es oligosaccharides of proteins delivered from the RER Proteins may be desIned for ER return Golgi lysosomes or secreIon out of the cell The Golgi apparatus is a stack of aOened membrane compartments with related compartments interconnected by a tubular network The size and number of stacks varies with cell type Cells that export lots of proteins pancreaIc cells have lots of Golgi The Golgi has ve disInct compartments Cis Golgi Network CGN site of delivery from ER Cis Golgi next region nearest the CGN Medial Golgi the central region Trans Golgi the distal region Trans Golgi Network TGN site of sorIng and exit of vesicles from Golgi network Measuring transport to the golgi by assessing changes in glycosidase sensi vity Protein runs faster in gel smaller when tree removed Oligosaccharide tree only sensiIve to experimentally added glycosidase if mannoses have been trimmed by enzymes in the cis golgi Proteins on the cytoplasmic face of vesicles regulate budding and fusion 1 Budding 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 Di erent vesicle types have di erent coat proteins COPII coats vesicles moving ER to Golgi COPI coats vesicles moving Golgi to ER and from more trans Golgi to more cis Golgi Clathrin coats vesicles moving from TGN to surface and from surface to endosomes lysosomes The contents of the vesicle apparently play no direct role in triggering budding Di erent coated vesicles serve speci c steps COP II subunits Di erent coated vesicles can be dis nguished morphologically A class of small G proteins regulates budding These cytoplasmic G proteins associate with membranes GTP bound budding is iniIated GDP bound no budding or removal of coat Di erent types of membrane budding use a di erent G protein COPII Sar1 COPI Clathrin Arf 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 GEF Non hydrolyzable analogs of GTP such as GTP s trap vesicles in the coated form The vesicle cargo Some soluble proteins seem to be carried along non speci cally by bulk ow Other soluble proteins seem to bind to cargo receptors which concentrate them in vesicles these transmembrane receptors interact via cytoplasmic domain with coat proteins Membrane proteins can themselves interact with coat proteins Implies a signal for export is needed to interact with coat Some proteins are speci cally 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 are AA sequences that interact with coat proteins Export signals in cargo receptors or membrane proteins themselves interact directly with the COPII coat proteins Budding Summary Sar1 acIvated COPII proteins begin to bud membrane Cargo loaded by bulk or by direct or receptor mediated interacIon with coat proteins Vesicle buds o COPII coat Sar1 hydrolyze GTP coat disassembles Now need to dock and fuse Yet another class of small G proteins help control docking to target membranes Docking and fusion are regulated by the Rab family of small Gproteins Di erent routes of vesicle tra cking appear to be controlled by di erent Rab family members over 60 Together with SNARES they control the speci city of vesicular tra c ER to CGN TGN to secretory endocytosis etc GTP bound state acIve Ightly associated with membranes or vesicles mediate tethering and docking of vesicles via a variety of e ector proteins in target membrane Hydrolysis to GDP allows release of inacIve Rab to cytosol Transmembrane proteins called SNAREs control fusion of the vesicle to the target membrane The coats disassemble ajer budding This means the coat does not play a role in targeIng Vesicles have transmembrane proteins called V SNARES These are recognized by T SNARES on the Target membrane provides direcIonality between compartments Bring membranes into close apposiIon allowing fusion Many unique T SNARE and V SNARE proteins exist and speci c pairings mediate speci c membrane fusion events RabGEF Transmembrane proteins called SNAREs control fusion of the vesicle to the target membrane The coats disassemble ajer budding This means the coat does not play a role in targeIng Vesicles have transmembrane proteins called V SNARES These are recognized by T SNARES on the Target membrane Bring membranes into close apposiIon