L3 - golgi-vacuole and endocytic Flashcards
what is the function of the vacuole/lysosome?
degradation of extracellular material taken in by endocytosis and degradation of intracellular compartments in autophagy.
what do lysosomes contain?
degradative/proteolytic enzymes (require certain PH) which need to be kept separate from the rest of the cell as they will begin to degrade the cell if not.
how do lysosomal resident enzymes transport into the lysosome?
they transport into the lysosome from the trans golgi via the secretory pathway
What dies VPS screen stand for?
vacuolar protein sorting screen
- if pathway blocked to vacuole does protein transport to PM instead?
what is CPY?
carboxypeptidase Y is an enzyme which is usually transported from the golgi to the lysosome (degradation)
how are cells which secrete CPY identified?
by a colour assay
- cells which secrete CPY (as go to PM instead of lysosome) are tested further and their genes are identified.
how many vacuolar sorting genes were identified?
63
how can western blot identify problem with reaching vacuole?
- if CPY reaches the vacuole part gets chopped off by golgi into the smaller from. (if no fast travelling band - no mature protein in vacuole)
what classes are vacuolar mutants divided into?
A- WT B- fragmented vacuoles C- no vacuoles D - large vacuoles E - vacuoles large F- large vacuoles surrounded by smaller vacuoles
what are the 4 possible destinations from the late golgi for a protein?
- lysosome/ vacuole
- plasma membrane
- to early endosome
- to late endosome
(sorting events determine where protein goes)
where is CPY usually transported?
to late endosome- then to lysosome?
(requires adaptor proteins and coat proteins
what gets sorted straight from golgi to vacuole?
- ALP (alkaline phosphatase), and VAM3 traffic from golgi to vacuole directly (bypass endosomes)
- ## adaptor protein AP3 required
what traffics to the early endosome, late endosome and back to the golgi?
Kex2
- enzyme which cleaves alpha
- transport to early endosome requires clathrin
- retention signals keep it in the golgi
- can be phosphorylated to move from early to late endosome
what is endocytosis?
the process where the PM invaginates into the cell resulting in the production of a vesicle which is able to fuse with endosomes to enter the endo-lysosomal membrane system.
- intake of components of EC environment?
why do cells need endocytosis?
- downregulation of signals (reduce EC signal level)
- retrieval of molecules that form part of secretory vesicle for recycling (can repeat process)
- remodelling of cell surface lipid and protein composition (can make fit for environment)
- entry into cell by toxins and pathogens
what are the stages in endocytosis?
- plasma membrane to endocytic vesicle
- endocytic vesicle to early endosome
- early endosome to late endosome/MVB (or recycling to PM)
- late endosome to golgi or lysosome/ vacuole
how do we know that there are multiple components and proteins dont just traffic straight from cell surface to the lysosome?
- see using microscopy different compartments
- mutants block specific stages of traffiking pathway
- early and late endosomes can be separated on density
(can follow sequential localisation of actin)
what does MVB stand for?
multivesicular body (late endosome in yearst)
- endosomes invaginate into membranes to form internal vesicles (intraluminal vesicles)
- MVB fuses with vacuole to deliver internal contents for degradation
how do we know sorting pathways into the MVB overlap?
- Ste3- localises to PM, is ubiquinated and internalised by endocytosis. Is traffiked from EE to LE/MVB to vacuole.
- carboxypeptidase S (CPS) traffiked from golgi to LE/MVB to the vacuole
- VPS10 - traffiked to LE/MVB and then retrieved to golgi
what is VPS10?
receptor for CPY
- Vps10 is retrieved to the late Golgi through a specific
aromatic -based signal in its protein sequence (YSSL, FYVF).
