MVU9- MEMBRANES - 4 Flashcards
where does the quality control for lumen proteins happen?
at the ER
where are misfolded secretory proteins degraded and by what system?
at the ER
ubiquitin proteasome system
need to go into the cytosol
what are the ER chaperones?
BiP (HSP70)
ERdj proteins (DNAJ co chaperones)
NEF co chaperones
GRP94 (HSP90) doesn’t have a co chaperone
thioredoxin family (PDI and ERp57)
which chaperone assists secretory protein folding?
substrate binding DNAJ (ERdj3)
what proteins does the translocon interact with during folding?
signal peptidase
OST
sec63 (co chaperone): specialised TM DNAJ that recruits BiP to translocating polypeptides (does not bind substrate directly)
how do disulfide spontaneously form in the ER lumen?
difficult, inefficient and/or incorrect
what proteins catalyse disulfide formation? and their structure
thioredoxins
PDI and ERp57
have 2 reactive cysteine residues close together, can oxidise the substrate
interact with the protein
how does PDI help the formation of disulfide bonds?
oxidized PDI catalyses the formation of that bond and becomes reduced
forms mixed disulfide intermediate with substrate
what is the pathway of PDI regeneration?
- PDI becomes reduced after oxidising the substrate
- PDI is oxidised by Ero1 with cofactor FAD, Ero1 is reduced
- Ero1 is regenerated by FAD
- FAD is regenerated by O2
oxidation potential in ER is maintained by controlled by enzymes (PDI, Ero1)
what is the structure of CNX (calnexin)?
50kDa lumenal domain and a TM helix anchor
what is the structure of CRT (calreticulin)?
has a lumenal domain, but with no TM helix, has a signal for retention in the ER
what do the CNX lumenal domain and CRT do?
recognise glycan pattern on polypeptides (N-linked glycosylation)
bind to thioredoxin (ERp57)
what does the N-linked glycan inform the cell about?
information about the stage of folding of the protein
3 glucoses: just came in
trimmed to one glucose: CNX and CRT will recognise it, signal for incomplete binding, but eventually removed
what does CNX bind to?
binds to the glycan with 1 glucose
keeps the polypeptide in the ER
what does UDP glucose do?
glycoprotein glucosyltransferase
binds non native polypeptides and reattaches a glucose to the glycan (CNX can bind it)
recognises hydrophobic patches
what does UGGT not recognise?
native folded polypeptides
what does glucosidase do?
removed Glc from native and non native polypeptides
what does mannosidase do?
trims sugar further, not recognised by UGGT
what is the calnexin and calreticulin cycle?
- CNX keeps polypeptide in ER
- glucosidase removes the last Glc
- UGGT restores Glc on misfolded polypeptides - CNX binding
- folded polypeptides do not have glc restored and exit to the GA
- mannosidase trims glycans without Glc
- slow, irreversible
- proteins retained in ER by chaperones likely to get trimmed - mannose binding lectins (EDEM) select short glycans for degradation
what does ER associated degradation (ERAD) degrade?
both lumenal and TM polypeptides
what are the steps of ERAD?
- substrates are recognised and brought to E3 Ub ligase complexes
- E3 complex polyUB the substrate, and retro translocated (from lumen to cytosol) substrate into cytosol
- substrate is deglycosylated and degraded by proteasomes
what is the first step of ERAD?
- Recognition by BiP
substrates which cannot fold are prevented from aggregating by BiP
BiP binds substrate in complex with specialised DNAJ (ERdj5) and lectin (EDEM)
ERdj5 has a J domain and thioredoxin domain, recognises BiP
it catalyses the breakage of the disulfide bond in the substrate
starts unfolding as much as possible
brings substrate to E3
what is the second step of ERAD?
transmembrane E3 ligases (HRD1 and gp78) form complexes with recognition adaptors:
misfolded lumenal proteins (SEL1L)
mannose binding lectins (EDEM)
misfolded TM proteins (erlin1/2, derlins)
chaperones (BiP)
other interactions with derlins and p97 receptors
explain retro translocation
still in question if retro translocation requires a true pore
regulated opening and closing
large enough to allow N-linked glycans
polyUb in the cytosol is necessary
large TM E3 ligase complexes are thought to perform all of those functions (independent from cytosol degradation):
- HRD1 and gp78 ligases: homologous, made up of multiple TM helices
retrotranslocation is assisted by the cytosolic protein p97/VCP
- pulling or unfolded activity
what is the p97 mechanism?
N linked glycans and polyUb are too large to fit through pore
p97 forms complexes with other proteins
- ub binding adaptors
- peptide: N-glycanase (PNGase) removes glycans (bc they are too big)
- DUBs remove poly-Ub
BUT E3s re Ub after extraction to complete targeting to proteasomes
what do AAA proteins do in this process?
large diverse super family of ATPases with many different functions
usually hexameric rings
unfoldase subunits of the proteasome 19S regulator
what does the p97 do and what is its structure?
homo hexamer of 97kDa subunits
uses ATPase energy to extract proteins from membrane
substrate is also threaded through the central pore
what is the summary of ERAD?
- Misfolded polypeptides are recognized by lectins and chaperones
- Disulfide bonds are broken by a thioredoxin DNAJ
- Adaptors bring misfolded polypeptides to E3 ligases
– lectins, chaperones, lumenal and TM adaptors - Transmembrane E3 ligases polyubiquitinate substrates and start
retro-translocation - p97 ATPase helps extract substrates from membrane
- Substrates are de-glycosylated (PNGase)
- Substrates are recognized by proteasome or shuttling receptors
what proteins undergo unfolded protein response?
ER proteins
what activates the UPR?
activated by accumulation of unfolded proteins in the ER
reductive stress: breaks in disulphide bonds
glycosylation inhibitors
loss of calcium
what is upregulated in UPR? and what can happen if the response is inefficient?
transcription of ER chaperones, ERAD components, lipid synthesis (allows the ER to expand in size)
can promote cell death if the response is insufficient (apoptosis)
what are the 3 UPR signaling pathways?
IRE1
PERK
ATF6
how does the IRE1 signaling pathway work?
IRE1 and XBP1
1. IRE1 has a lumenal domain, kinase
and RNase domains
2. IRE1 dimerizes in response to
unfolded proteins
3. Autophosphorylation activates RNase
activity
4. XBP1u (unspliced) is translated at
very low levels
5. IRE1 splices out 26 base intron,
frameshift allows XBP1s (spliced) to
be translated efficiently
6. XBP1s is a transcription factor that
upregulates UPR genes (more chaperones, ERAD proteins and lipid synthesis)
how is IRE1 activated?
- Direct binding of unfolded protein by
2 IRE1 proteins causes dimerization - BiP (HSP70) binds inactive IRE1 and
prevents dimerization - BiP binding to unfolded protein releases
IRE1 to form dimers - Both mechanisms activate IRE1
how does the PERK mechanism work?
- PERK has a lumenal and a kinase domain
- PERK dimerizes upon stress and
autophosphorylates
– same activation mechanism as IRE1 - Phosphorylates translation factor to inhibit
translation - Certain mRNAs are not inhibited:
– ATF4 transcription factor - ATF4:
– expression of more XBP1
– CHOP – activates apoptosis genes
– decision: recover from stress or commit
to cell death
how does the integrated stress response work?
- Translation initiation factor eIF2α turns on
general translation - Phosphorylated eIF2α is inactive
- eIF2α Kinases respond to different stresses to
inhibit translation
– decrease amounts of unfolded new
proteins
– special mRNAs including ATF4 are still
translated to promote cell death - PERK – ER stress
- Other kinases – proteasome inhibition (HSR)
starvation, viral infection, etc
hoes does the ATF6 pathway work?
- ATF6 is normally transmembrane at the ER –
BiP binding site masks ER exit signal - Upon stress, BiP is competed away by unfolded
proteins - ATF6 is transported to Golgi
- Golgi proteases cleave off cytosolic domain
ATF6(N) - ATF6(N) is a soluble transcription that upregulates
UPR genes
