Vesicle Transport

• budding: sorting complex formation, for forward or retrieval
• transport: movement of vesicle to acceptor
• docking: specific pairing between fusion partners
• fusion: at PM called exocytosis: may be regulated or constitutive

Vesicles

• 50-75nm diameter
• coated during budding
• mechanical force for budding
• selecting membrane components (and thus cargo)

Sorting Mechanisms

• retention= exclusion from transport vesicles
• TM domain
• size of complex/ anchored to cytoskeleton
• displacement?
• retrieval=> retrograde flow
• signal for coat inclusion
• KDEL for lumenal proteins, bind KDEL receptor
• KKXX on cyto tail for membrane proteins

COPII (ER to Golgi)

• exchange of GDP for GTP on Sar
• sarGTP binds membrane
• recruitment of CopII complex (sec23,24,13,31)
• recruitment of cargo bound sorting receptors thru coat binding
• sarGTP hydrolysis (coat is GAP)
• coat disassembly

COPI (Golgi to ER, ER to Golgi thru Golgi,inter-endosome)

• exchange of GDP for GTP on Arf
• ArfGTP membrane binding
• recruitment of COPI (alpha, beta, gamma etc.)
• recruitment of cargo bound sorting receptors
• ArfGTP hydrolysis
• coat disassembly

Clathrin (TGN to endo, PM to endo, from endo or lyso to ?)

• outer clathrin coat (triskelians)
• inner adapter complexes: bind clathrin and tails of membrane receptors
• receptors bind cargo

Example of targeting is lysosomal hydrolase sorting

• N-linked CHO addition and delivery to Golgi
• NAG phosphotransferase + phosphodiester gycosidase phosph. in cis Golgi
• M6PR (large and small) binding in TGN (pH 6.5)
• binding of cyto tail to AP1/clathrin
• budding
• movement and fusion with LE
• MPR dissociation from ligand (low pH 5.5-6.0)
• MPR retrieval (uncoated vesicles?)

Docking

• binding partners in vesicle or target membrane
• specificity achieved by layers of these interactions
• for simplicity we will just consider SNAREs which also mediate fusion
• v-SNAREs are family of C-terminally anchored proteins
• incorportated into vesicles by interaction with coat
• must be retrieved after fusion
• note that during retrieval they should be inactivated

t-SNAREs are also family of C-terminally anchored proteins

• interact with SNAP25 (lipid anchored membrane protein)
• label target membrane for specific v-SNARE
• v/t SNARE pairing (ie binding) leads to docking
• note that complex must be broken after fusion

Fusion

• must overcome significant energy barrier for lipid bilayer fusion
• steps involve (see diagram):
• dimple formation
• hemifusion intermediate
• fusion pore formation
• thought to be mediated by transition of v/t SNARE pair to core complex
• very tight binding=> highly favored
• twisted four stranded helical bundle (see diagram)
• brings membranes into close apposition for lipid rearrangements
• note that core has to be disrupted after fusion

Recycling

• to break v/t SNARE pair which is now a core complex in target membrane
• need to input energy
• achieved by NSF ATPase
• large multi-subunit molecule with central cavity
• may sit on core complex and use ATP driven conformation change to
• disrupt core
• also requires cofactors for association with SNAREs, called SNAPs
• V-SNARE can now interact w/ a retrieval sorting complex (ie coat)

Regulation

• constitutive fusion is actually regulated
• in the sense that the overall rate is determined by rabs
• rabs are a family of low MW GTPases controlled by GAPs and GEFS
• prior to docking vesicles acquire rabGTP
• GEF needed to exchange GTP for GDP and allow membrane binding
• upon fusion GAP activates GTP hydrolyis-> rabGDP dissociates
• mechanism of regulation is unknown
• regulated fusion is Ca++ dependent
• Ca++ may bind a Ca-binding inhibitor of core complex formation
• once calcium binds inhibitor it dissociates and fusion progresses