Lecture 17: The Endocytic/Lysosomal Pathway Flashcards
Explain what endosomal sorting it
They are the destination of endocytic vesicles from the PM or TGN and proteins can either be recylced back to the PM or targetted to the lysosome.
How are MVBs formed? What is this called and what is it mediated by?
when the membrane of the endosome buds into the lumen forming intracellular vesicles loaded with cargo. the point of no return. ESCRT machinary.
Which pathway is used by receptors, toxins, viruses, bacteria and circulating proteins?
Receptors use clathrin dependent endocytosis, the other use caveolin endocytosis
How are integrins internalised?
Using ARF GTPase
What happens to monoubiquitinated proteins in the early endosome?
They are recognised by proteins with ubiquitin binding domains and there is envagination of the endosome membrane to form the MVB that target the protein to the lysosomes.
Where are proteins stored that are destined for the lysosome membrane not lumen?
The MVB membrane
What are Rabs and what do they do?
They are members of the Ras superfamily and they regulate membrane trafficking such as vesicle formation and membrane fusion
Explain how ubiquitin modified cargoes are recognised and sorted into endocytic vesicles
they are recognised by proteins with ubiquitin binding domains and ubiquitin interacting motifs. Ub can be used as a signal for internalisation and is required for inclusion into MVBs. ESCRT proteins recognise the Ub and promote the invagination of the endosome membrane to form MVBs.
Give an example of ub protein being sorted.
Cbl ub ligase puts Ub on the rtk protein and epsins bridge the rtk to clathrins. ESCRT proteins help dock the rtk onto the endosome
What is recognition of Ub mediated by at endosomes?
ESCRT-0
Which proteins does cargo sorting at endosomes require? What else are they needed for?
ESCRT-0, 1, 2, 3 and Vps4 AAA-ATPase. MVB formation.
Describe the structure of ESCRT-0.
Heterodimer of Hrs and STAM1/2 subunits in a 1:1 ratio.
What is the main difference between the two domains?
Hrs has FYVE domain which enables ESCRT-0 to bind PI3P. Hrs also engages clathrin.
What are the four ubiquitin binding domains in ESCRT-0?
DUIM, VHS from Hrs and VGS and UIM from STAM.
What are the roles of ESCRT-0 and epsins? What is similar about them?
ESCRT-0 for recognition of Ub, epsin for sorting of cargoes on the PM.Similar structure with multiple UBDs, lipid binding domains and interface for interaction with vesicular machinery.
Explain how ESCRT-0 docks at endosomes.
Both subunits bind Ub so bind the Ub cargo. PI3P binding is also crucial. ESCRT-1 interacts with ESCRT-0 via the Hrs C-terminal.
What is different in ESCRT-1 to 0?
1 only weakly binds PI3P
Describe the structure of ESCRT-1.
has flexible link connecting the ubiquitin binding domains. Several UBDs incljding glue and uev.
What is the structure of ESCRT-2?
Y shaped heterodimer with one subunit of Vps22, 36 and two subunits of Vps25.
How does ESCRT-2 interact with ESCRT-1?
via their GLUE domains.
What ensures endosomal localisation and how does ESCRT-2 bind this?
Binding of pi3p and through the GLUE domain.
What do all ubiquitin binding domains recognise?
IL44 patch on ubiquitin
What doesn’t ESCRT-3 have?
A ubiquitin binding domain
What happens if you remove individual UBDs in ESRCT-1 compared to all 3 in ESCR-1 and 2?
Nothing happens compared to sorting of cargo into ILVs is prevented.
What are the subunits of ESCRT-3?
CHMP6,4,3,2
What does ESCRT-3 do and what happens to it?
It wraps around the necks of forming ILVs to promote their scission and ESCRT3 is then disassembled and recycled by Vps4.
What happens once ESCRT3 is assembled?
It needs energy to disassociate from the membrane which it gets from Vps4 ATPase
What is Vps4 and what structure is it?
It is a multimeric mechanoenzyme that binds ESCRT3 via N terminal MIT domain. It is a dodecamer when assembled.
What are ILVs?
Intra-lumenal vesicles
Where else could ESCRTs be important?
Autophagy because you need to close the vesicles still.
What does sorting cytokinesis and virus budding require? What do defects result in?
interaction of ESCRT3 with ALIX. Alix mutant deficient for CHMP-4 binding leads to cytokinesis defect and HIV-1 budding failure.
What does release of HIV-1 require and what recruits this?
specific lipids and proteins which are recruited to the budding site by gag.
What does Gag do?
Binds the PM via n terminal and binds ESCRT1 and ALIX via C terminus.
