Transport II Flashcards
Mechanism for vesicle budding
-Sec 12 converts sar1 GDP to sar1 gtp
-sar1gtp inserts into membrane
-sec23/24 bind and deform membrane and recruit cargo= coating bud
-budding
-gtp hydrolysis by sar1 and coat disassembles
-ready for fusion
-
Which proteins are sufficient for vesicles budding?
From the ER to the Golgi,
sar1 (gtp binding protein)
Sec 12 (GEF)
Sec23 and sec24 ( coating proteins)
Sufficient
Example is seeing if NLS is sufficient for nuclear translocation…add the 8AA to GFP and see if the nucleus lights up.
Necessary
Transfect plasmid with GFP hooked up to mutant NLS and see that compared to WT it doesn’t enter nucleus.
Mechanism of vesicle fusion
V snares (vesicle) and t snares (target) make complex to initiate fusion
Vesicle Fusion proteins
SNARES: families of shot helical TM proteins that allow for fusion specificity
Specificity comes from snares and rab proteins (small GTPases)
Vesicle fusion at PM. Proteins and mech
Proteins:
- VAMP = v snare
- syntaxin & SNAP 25 = t snares
- rab = small GtPase and binds rab effector on PM
- NSF = ATPase that dissociates SNARE complex
Mech:
- rab GtPase on vesicle binds to rab effector on PM
- v+t snare complex forms, brings membranes closer together
- fusion
- NSF is an ATPase that dissociates snare complex and release contents from vesicle to membrane
Endocytosis mech
PM➡️early endosome ➡️ late endosome➡️lysosome
Exocytosis mech
ER➡️Golgi ➡️PM
Experiment to find exocytic pathway
Old approach: pulse with radio labeled leucine for given time
Chase with non radio labeled leucine at different times
Autoradiography to determine location of radioactive proteins
New approach: hook GFP onto temperature sensitive mutants of secreted protein. At different times, put at sensitive temp or good temp. Sensitive temp (40) stopped process and allowed them to view where fluorescence. Put at good temp for given time and then 40 again to see the progression. Found protein went from ER ➡️Golgi ➡️PM
Three ways for proteins to cross membranes
- Gated transport (nuclear pores)
- Protein translocation (ER)
- Vesicle transport (budding and fusion)
