Module 4 Flashcards
Protein Trafficking & Vesicular
Transport within the secretory
system of cells.
- Which efficiently delivers and regulates the levels of over 5,000 transmembrane proteins and 2,000 secreted proteins in humans.
- Correctly locates each protein within the cell to allow it to function
- Dynamically moves proteins within the cell as required
Anterograde traffic pathway
Secretory Pathway/Exocytosis
Retrograde trafficking pathway
Endocytosis/Uptake
Temperature Sensitive O45 variant of
the vesicular stomatitis virus G
- mutant has temperature sensitive trigger so at a particular temperature it will remain unfolded
- this means the chaperones in the ER will not release it and let it fold correctly
- Raise the temperature (to a certain degree) the molecule folds correctly and is transported normally
Intracellular compartments
Donor compartment vs Target Compartment
Transport Vesicles
come in different flavors
COP1
COP2
CLATHRIN
Donor to target compartment transport
- Budding
- Scission
- Uncoating
- Translocation
- Tethering
- SNARE assembly
- Fusion
- in donor compartment: soluble cargo and transmembrane cargo that need to be in transport vesicles (need to be directed for this to happen)
- protein coat binds to cargo and deforms membrane
- this membrane bending is energetically unfavourable to cell
- once everything inside its a budded vesicle and undergoes cission
- it fuses with membrane on target side and releases it
- coat proteins are disassembled and reused
ER protein trafficking
transmembrane and secreted proteins from ER to cis-golgi, via COPII vesicles (anterograde)
vice versa COP1 vesicles
COP II dependent vesicular transport
initiation:
- GDP-GTP exchange on Sar I catalysed by the transmembrane guanine nucleotide exchange factor Sec12.
- Activated Sar1-GTP binds to membrane.
coast recruitment and assembly:
- Sec 23/24 recruited and Sec24 captures exposed transmembrane cargo sigal (prebudding complex which are clustered by Sec13/31 generating COP II-coated vesicles.
coast disassembly:
- Sec23 promotes hydrolysis of GTP by Sar1. This promotes the release of Sar1-GDP causing disassembly [ Sec12: GEF, Sec23: GAP]
retrieval:
- most ER resident proteins have sorting signals to bind to KDEL receptors or COPI coat directly
Targeting
tethers are peripheral membrane proteins and often interact w/ small GTPase proteins in vesicle or acceptor membrane. Enables SNARE
Mechanisms of vesicle fusion
- Vesicle Docking
- Rab-GTP on vesicle engages a Rab effector and GTP is converted to GDP during fusion process - Assembly of SNARE Complexes
- VAMP(v-s) interacts with SNAP-25 and Syntaxin(t-s) holding vesicle in place.
[ 2x SNAP, 1x VAMP, 1x syn helices] - Membrane Fusion and SNARE Disassembly
- NSF and alpha-SNAP bind to complex which disassociates the complex.
Exocytosis
TGN-Transport Pathway: regulated, constitutive, to endosome, mature within golgi
Constitutive Secretion: sorting signal independent, secreted at constant level
Regulative Secretion: level of secretion dependent on signals at constant level
Insulin: secreted from beta cells in the pancreas in response to high glucose concentrations in blood
Formation of mannose 6-phosphate (M6P)
phosphotransferase binds lysosomal hydrolase at signal patch and UDP-GICNAc. UMP removed creating complex that is released. GlcNac then removed leaving M6P
M6P targets proteins from Golgi to lysosome
addition of M6P to lysosomal enzymes in cis-golgi. M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicle. Dissociation occurs in early endosome due to acidic pH. Phosphate removed from endosome and M6P receptor retrieved back to trans GN via retromer-mediated sorting
pM-TGN-Lysosome Pathway M6P and M6PR
M6PR can be required at plasma membrane in case accidentally constitutively secreted. Then returned to endosome and eventually lysosome through endocytosis
