Quiz 2 Material Endomembrane System ER Protein Import ER Protein Folding Intracellular Vesicle Transport Golgi Endocytosis Lysosomes Exocytosis Intracellular Vesicle Transport How Do Vesicles Get to Their Des2na2ons How cid 3 do cid 3 vesicles cid 3 get cid 3 to cid 3 their cid 3 destinations CBIO3400 DATA cytoskeleton provides tracks motors to move vesicles tracks MTs acBn motors kinesin dynein myosin control Rab e ectors link vesicles organelles to motors The cid 3 cytoskeleton cid 3 provides cid 3 tracks cid 3 and cid 3 motors cid 3 to cid 3 move cid 3 vesicles tracks Microtubules cid 3 Actin cid 3 Motors Kinesin cid 3 Dynein cid 3 Myosin Control Rab Effectors cid 3 link cid 3 vesicles organelles cid 3 to cid 3 motors DATA CBIO3400 Budding cid 3 yeast cid 3 Saccharomyces cid 3 cerevisiae cid 3 as cid 3 a cid 3 model cid 3 for cid 3 the cid 3 analysis cid 3 of cid 3 intracellular cid 3 transport Budding Yeast as a Model for Analysis of Intracellular Transport experiment create temp EsensiBve mutants that can be turned o just by raising temp to a restricBve temp where protein cannot fold or funcBon properly ER cid 3 exit cid 3 sites cid 3 green cid 3 cid 3 and cid 3 Golgi cid 3 red transport into ER budding of vesicles from ER fusion to Golgi vesicles transport from Golgi secretory vesicles transport of secretory vesicles cell surface Generic Vesicular Transport Process 1 Select site for vesicle formaBon 2 Assemble coat 3 Select load cargoes 4 Pinch o vesicles 5 Uncoat vesicles 6 Direct vesicle to target membrane 7 Fuse vesicle w target membrane Types of Coats DATA Types cid 3 of cid 3 coats cid 3 CBIO3400 COP1 COP2 clathrin retromer Golgi cid 3 to cid 3 ER cid 3 Golgi cid 3 to cid 3 Golgi cid 3 e g cid 3 cis to cid 3 trans ER cid 3 to cid 3 Golgi to cid 3 and cid 3 from cid 3 plasma cid 3 membrane endosome endosome cid 3 to cid 3 Golgi COP1 Golgi ER transEGolgi cisEGolgi moves BACKWARD COP2 ER Golgi moves FORWARD clathrin plasma membrane endosome transEGolgi retromer endosome Golgi Coat Proteins COPs COP1 complex Golgi ER transEGolgi cisEGolgi BACKWARD 7 subunits ADP RibosylaBon Factor ARF regulates COP1 complex COP2 complex ER Golgi FORWARD subunits Sec23p Sec24p Sec13p Sec31p form dimers Sar1p related to ARF Sec12p GEF for Sar1p ADP Ribosyla2on Factor ARF1 Sar1p slide 1 of 5 1 member of GTPEbinding protein family 2 myristoylated NEterminus important for funcBon myristoyla2on when FAs are added to protein ARF is myristoylated in GTPEbound form FA hidden inside protein binds GTP myrisBc acid gets to surface allows protein to stably anchor itself to membrane myrisBc acid is extended into membrane from protein when protein is in GTPEbound state coEtranslaBonal modi caBon EX many GEprotein opsin DATA ADP Ribosyla2on Factor ARF1 Sar1p slide 2 of 5 The cid 3 Arf GTP cid 882 GDP cid 3 cycle CBIO3400 3 helps de ne sites of vesicle formaBon ARF GDP cytosol GEF membraneEbound acBvates ARF ARF GTP can interact w membrane membraneEbound coat proteins recruits COPs GAP GEF INACTIVE cytosol membraneEbound ACTIVE GAP cid 3 GTPase activating cid 3 protein GEF cid 3 cid 882 guanine cid 3 nucleotide cid 3 exchange cid 3 factor in cid 3 membrane ADP Ribosyla2on Factor ARF1 Sar1p slide 3 of 5 3 helps de ne sites of vesicle formaBon DATA ARF GTP recruits COPs ARF1 cid 882 GTP cid 3 recruits cid 3 heptameric coat cid 3 complexes CBIO3400 can make condiBons nice for vesicle formaBon coatomer aJracted to GTP acBvated state vesicle pinches o coatomer is stripped COP1 coat brings proteins leaked into Golgi from ER back to ER receptors leave ER w cargo vesicles go to Golgi receptors brought back to ER in COP1Ecoated vesicles Arf1 cid 882 GEF A R F G D P A R F G E F A R F G T P c o a t o m e r ADP Ribosyla2on Factor ARF1 Sar1p slide 4 of 5 Arf cid 882 effectors cid 3 remodel cid 3 the cid 3 membrane DATA CBIO3400 4 indirectly changes membrane lipid composiBon via e ectors FEBS cid 3 Letters cid 3 Volume cid 3 583 cid 3 Issue cid 3 23 cid 3 2009 cid 3 3872 cid 3 cid 882 3879 acBvates PCEspeci c phospholipase D increases phosphaBdic acid in microenvironment modi caBons made by ARF GTP negaBve curvatures reduce size of polar head groups posiBve curvature one FA chain removed from lipid Lyso cid 882 lipids PA cid 3 DAG ADP Ribosyla2on Factor ARF1 Sar1p slide 5 of 5 5 controls uncoaBng of vesicles ARF GAP has 2 domains that sense curvature of membranes ALPS mo2fs ARF GAP cannot bind planar membrane only curved membrane means ARF GAP is aJracted to where vesicle forms to vesicle itself will switch o ARF GTP stop process does this via ALPS mo2fs ALPS mo2f amphipathic Ehelix rich in S T on one side hydrolysis of ARF s GTP leads to uncoaBng of vesicle Intracellular Vesicle Transport Steps 1 ARF GDP is present 2 ARF GEF comes along exchanges GDP for GTP 3 ARF extends its myrisBc acid from within 4 ARF binds itself to membrane 5 This aJracts the coatomer 6 Coatomer interacts w TM domain of cargoes 7 This aJracts cargo to site As long as there s enough ARF GTP present vesicle coat will grow cargo will be aGracted to vesicle by coatomer 8 Vesicle pinches o 9 GAP induces GTP hydrolysis ARF returns to its GDPEbound state Coat gets destabilized is essenBally removed from vesicle DATA CBIO3400 SNAREs SNAP cid 3 Soluble cid 3 NSF cid 3 Attachment cid 3 Protein cid 3 REceptor cid 882 Monomeric cid 3 v cid 882 SNARE cid 3 synaptobrevin etc cid 882 Trimeric t cid 882 SNARE cid 3 syntaxin cid 3 Snap25 cid 3 etc cid 882 Many cid 3 35 cid 3 in cid 3 animals cid 882 Bring cid 3 membrane cid 3 of cid 3 V cid 3 and cid 3 T cid 3 close cid 3 together cid 3 1 5 cid 3 nm v cid 882 and cid 3 t cid 882 SNARES cid 3 are cid 3 complementary cid 3 sets cid 882 cid 882 Targets cid 3 of cid 3 tetanus cid 3 and cid 3 botulism cid 3 neurotoxins cid 3 SNARE cid 3 specific cid 3 proteases DATA CBIO3400 SNAREs Figure 13 16 Molecular Biology of the Cell Garland Science 2008 SNARE cid 3 function v cid 882 and cid 3 t cid 882 snares cid 3 from cid 3 trans cid 882 SNARE cid 3 complex tetrameric coiled cid 882 coil zipper cid 882 mechanism cid 3 can cid 3 be cid 3 delayed cid 882 cid 882 make sure vesicles nd target membranes cid 882 proteins in vesicles on membranes cid 882 Controlled cid 3 accessibility cid 3 RABs cid 3 etc important for recognizing correct target vesicle fusion allows membranes to come very close together H2O …