Lecture 16: Membrane Protein Turnover Flashcards
What happens to proteins that are destined for the ER, Golgi, PM and lysosome?
Cotranslationally inserted into the lipid bilayer of the ER
What two DELIVERY pathways does Ub regulate?
delivery of proteins to the PM in the secretory pathway and the removal of proteins from the PM via the endocytic pathway.
What ways can Ub target proteins?
For the lysosomal pathway and for ERAD/ proteosomal degredation.
How long is Ub, how is it assembled into chains and what residues can it be assembled to?
76 AAs, assembled between the c terminus of one Ub and the N terminus of any of the seven lysines of another Ub. K6, 11, 27, 29, 33, 48, 63.
Where is the isopeptide bond formed?
between the NH2 group of a lysine on a protein and the C terminal of G76.
What are some of the lysines for?
K6 for DNA repair, 11 for cell cycle, 27 for mitophagy, 48 for UPS and 63 for autophagy.
What are E1, 2 and 3 for and what removes Ub. How many of each exist?
E1 activating enzyme,2, E2 conjugating enzyme (37) and E3 ub ligase (>600) and deubiquitinase (around 85)
What is the problem with integral membrane proteins and how is this overcome?
Proteosome can’t access them. So controlled by membrane trafficking that is governed by monoub and K63 polyub for autophagy to target proteins to the lysosome and k48 ub chains for ERAD.
Describe the lysosomal pathway
There is invagination of the PM, the clathrin coat is important for structure, there is vesicle trafficking through early endosome, multivesicular bodies before delivery to lysosome
What can the lysosome do?
be expanded due to a dynamic PM and can engulf mitochondrias
Compare the lysosomal and UPS systems.
Lysosomal: Ub is added, protein taken to endosome then MVBF then lysosome where membrane fission occurs and will be used if a protein has made it all the way to the PM.
UPS: E1, E2 add Ub, E3 takes E2 with bound protein and adds Ub chain before sending to 26S proteosome for degredation. Mostly k48 chains and the proteosome has lots of proteases. mostly cytosolic and ER proteins.
Explain how ER associated degradation is carried out.
Proteins without an ER retention signal (KDEL for lumen bound, KKXX for TM) are transported in COP2 vesicles to the golgi. Proteins that do have an ER retention signal are retained in the ER, usually for proper folding. Ub doesn’t seem to be involved in ER exit. Proteins that are misfolded undergo ERAD.
What do UBX proteins have?
Ubiquitin binding domain (UBD) for substrate recognition and UBX domain for recruitment of AAA ATPase CDC48/p97 necessary for membrane extraction.
What happens after UBX proteins have bound to the protein target?
It is fed through the ER (retrotranslocated)
What is the role of herp and BiP in ERAD?
Herp facilitates oligomerisation of the Hrd1 E3 ligase. BiP binds to a number of glycosylated and non-glycosylated ERAD substrates and provides a barrier to the ER luminal side of the translocon.
What does hrd1 do?
Ubiquitinates the polypeptide chain.
What are the components of the retrotranslocon? What does this recruit?
Derlin, Hrd1 with its partner Sel1 which recruits the cytosolic ATPase p97
What does p97 function as?
A complex with cofactors that have UBX domains eg UBXD1 and UBXD7.
What are p97’s partner proteins?
Ufd1 and Npl4
What happens to the retrotranslocon once the substrate has been moved through?
It can be disassembled before a new substrate engages
What has to happen to the proteins before they go through the proteosome?
They have their carbohydrate and ub chains removed
What does calnexin do?
Acts as a chaperone ti retain unfolded or unassembled N-linked glycoproteins in the ER.
Why is ERAD so important. What pathogen hijacks it and why?
ERAD prevents the escape of misfolded proteins into the secretory pathway. Cytomegalovirus prevents surface expression of key immune response regulators including histocompatibility complex class 1.
In yeast what is the e3 ubiquitin ligase in golgi
Rsp5
What mediates golgi to endosome traffic?
GGAs
What do GGAs contain?
4 domains, VHS, GAT, gamma adaptin and a hinge region that bind clathrin.
What do GGAs do?
Recognise dileucine motifs in cargo via the VHS domain or by the GAT domain binding ubiquitin. They sort the proteins into clathrin coated vesicles that traffic directly to endosomes.
What do GGAs bind at the golgi complex?
PI4P
What does disruption of GGA function do?
results in aberrant trafficking phenotypes in both yeast and mammalian cells
What could an additional function of ubiquitin dependent sorting at the golgi be?
Be part of the quality control which degrades unfolded TM proteins that have escaped the quality control system at the ER.
What can happen with or without Ub?
The internalisation of proteins by endocytosis eg of RTKs and GPCRs
What is there a competition between?
Ubiquitin and deubiquitin activities
How are ub-modified cargoes sorted into endocytic vesicles?
Epsins interact with Ub and clathrin.
What is the destination of endocytic vesicles?
Endosomes
Where do endocytic vesicles come from?
The PM or TGN
What happens at the endosomes?
Some proteins are targetted back to the PM and some to the lysosome
Give an example of ubiquitin mediated endocytosis.
Downregulation of epithelial Na channels is Ub dependent and important for iron homeostasis in EC cells.
