BIOC 212 Lecture Notes - Lecture 11: Feedback, Apoptosis, Major Histocompatibility Complex
28 May 2018
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7- Membrane Proteins
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Retro-Translocation
• Still in question if retro-translocation requires a true pore
o Still researched; not entirely sure how proteins get retrotranslocated
• Probably requires a pore, just like translocation
• Can move out all kinds of proteins (TM, lumenal, etc.)
o Regulated opening & closing is pore-like
o But large enough to allow N-linked glycans
• If there is a pore, it must be large enough to allow the glycans to go
through, since cannot be removed until protein gets degraded
• Poly-ubiquitination in the cytosol is necessary for retro-translocation
o But poly-Ub is only put on in the cytosol
• Large transmembrane E3 ligase complexes are thought to perform all of these
functions at the same time
o Start retrotranslocation, have pore-like activity, and as bring polypeptide
across start polyubiquitinating them in the cytosol
o HRD1 & gp78 E3 ligases: homologous, multiple TM helices
• In addition to E3 ligases, gave multiple TM helices which suggest that
can act as a pore
o Derlins are associated TM proteins, may help to form pore
• Retro-translocation is assisted by the cytosolic protein p97/VCP
o p97 is not a membrane protein (in cytosol), but helps pull the protein out of
the ER during the retrotranslocation process
o Pulling or unfolding activity
• E3 ligases (N-end rule, CHIP, SCF E3 ligases) are only in cytosol
o Soluble protein
o E3 ligases evolved in ERAD are specific type that are membrane-anchored
at the ER
HRD1 & gp78 E3 Ligases
• HRD1 & gp78 (E3 ligases) form complexes with substrate recognition adaptors
o Cross the membrane, and have elements on the lumenal sides that recognize
different features of denatured proteins
• E3 ligase activity is on the cytosolic side
o Work with adaptor proteins that help bring various sets of polypeptides to the
E3 for degradation
• Different adaptors that recognize:
1. Misfolded lumenal proteins (SEL1L)
2. Mannose-binding lectins (OS9, XTP3, EDEMs)
• Recognize short mannose sequences
• Have binding site for ERdj5
• Have another set of adaptors which are integrated into the membrane
3. Misfolded TM proteins (erlin1/2)
• Recognize and bring them sideways into the E3 ligase
4. Chaperones (BiP)
• Derlins: proteins that help form the pore
o Interact with the E3 ligases
• p97 adaptors (UBXD8, VIMP)
o P97 in the cytosol helps pull polypeptide through the pore to reach the
proteasome (cytosol)
o Other proteins that temporarily hold p97 to the E3 ligase pore (adaptors)
Derlins
• Derlins (1 to 3) have 6 TM helices
• Important for retro-translocation
• Part of a larger family of proteins, most of which is made of proteases
o Derlins are not proteases, but sequence similarity implies some kind of
structure
• Proteases have one part which can open up, bind the polypeptide, close
and cut it
• Homologous to proteases that open up and cleave inside TM helices
o Derlins may open up the same way to form pore, or part of pore
• Instead of cutting polypeptide, open to form pore
P97 & AAA Proteins
• Protein in the cytosol that helps pull proteins out of the ER
• AAA-proteins
o Large, diverse superfamily of ATPases with many different functions in cell
o Unfolding activity
o Usually hexameric rings
o AAA for ATPases associated with activities
• Unfoldase base subunits of the proteasome 19s regulator is also part of the AAA-
protein family
• p97: homo-hexamer of 97 kDa subunits
o Uses ATPase energy to extract proteins from membrane
o Ub-binding adaptors (Ufd1, Npl4) needed for retro-translocation
§ Recognize the poly-Ub that E3 ligases have put on
§ Grab the Ub and help pull things out
o Unlike 19s unfoldase, substrate may not go through central hole
§ The pore of p97 is mostly blocked so substrate probably does not go
through the central hole like for proteasome
Document Summary
Interact with the e3 ligases: p97 adaptors (ubxd8, vimp, p97 in the cytosol helps pull polypeptide through the pore to reach the proteasome (cytosol, other proteins that temporarily hold p97 to the e3 ligase pore (adaptors) Derlins: derlins (1 to 3) have 6 tm helices. Instead of cutting polypeptide, open to form pore. Recognize the poly-ub that e3 ligases have put on. Grab the ub and help pull things out: unlike 19s unfoldase, substrate may not go through central hole. N-linked glycans are too large to fit into proteasome, but can still be retro-translocated. Enzyme that removes the glycan chain from the polypeptide. In lumen, break disulfide bonds using erdj5, but do not remove the glycans until are in cytosol, working with p97. N-linked glycan chains can pass through the retrotranslocation pore (e3s), but too large to fit into proteasome: ub extrinsic receptors (hr23a, plic1) Soluble proteins will bind to ubiquitin and bring them to proteasome.
