30 – Protein Translocation & Folding in ER Flashcards
Sec61 complex function
= ER translocator in all eukaryotic cells
o From cytosol to lumen of ER
what is Sec61
protein complex with 3 subunit = form a channel within ER membrane
Sec61 Exist in…
exist in all eukaryotic cells from yeasts to humans
-Highly conserved
Elongating polypeptide passes through Sec61 based channel with …
close contact with the ⍺ subunit of Sec61
Crosslinking agent makes the polypeptide stay in the channel
Discovery of Sec 61
Identified Sec61 through genetic screen in yeasts
-through secretion mutant screen of class A mutant where protein are accumulated in cytosol
23 proteins require for translocation- From ER to extracellular space
Structure & action of Sec61
Side view:
Short helical peptide acts as plug to close channel in absence of polypeptide to prevent passage of ions & small molecules
-Plug in place
Structure & action of Sec61
Top view:
Ring of isoleucine act as gasket to prevent leakage of ions & small molecules when polypeptide is translocated
-The plug moves away for some time when polypeptide is translocated
Lateral exit to lipid bilayer
* Exit seam
Protein folding in lumen of ER
Co-translational
need to fold polypeptide once translocated into ER lumen
Molecular chaperone BiP
BiP: Binding of immunoglobulin (Ab) Protein – ATPase, exists in lumen of ER in all eukaryotes
Conserved for folding
how does BiP binding work? step by step
- ATP-BiP hydrolyzed to ADP-BiP by Sec63 = pulls nascent polypeptide into ER lumen
-(Act as a ratchet) - ADP-BiP binds to new polypeptide chain
- Bound ADP-BiP prevent non-specific aggregation of nascent polypeptides in ER lumen
-to be folded correctly - Once polypeptide is folded correctly = ADP-BiP are phosphorylated back to BiP-ATP & released from polypeptide
2 important function of BiP
act as ratchet - to pull polypeptide into ER lumen
to prevent non-specific aggregation of polypeptide
N-linked glycosylation
Almost all proteins made in ER = N-glycosylated in highly ordered way in ER
N-linked glycosylation schematic step by step
- (Glc)3(Nan)9(GlcNac)2 added to selected Asn(N) in protein
- 1st glucose trimmed by glucosidase I
-ER localized - 2nd glucose trimmed by glucosidase II
- 3rd glucose trimmed by glucosidase II
- 2 options:
-New glucose added back by UGGT (glucosyl-transferase)
OR
-Mannose trimmed by mannosidase
-protein correctly folded
Cells use N-glycosylation to
to aid protein folding in ER
Tag to tell which polypeptide is properly folded
chaperone protein for folding for N-glycosylation
-Calnexin (CNX) and/or Calreticulin (CRT)
Cells using N-glycosylation: step by step:
- After 1st two glucose are moved, third glucose is recognized by chaperone proteins for folding
-Incompletely folded protein is trapped
-Off-pathway aggregation prevented - After some time, the 3rd Glc is remove by glucosidase II = releases protein from CNX/CRT
- If folded correctly = mannose is trimmed by ER mannosidase to produce (Man)8(GlcNAc)2 for Golgi transport
OR
-If not properly folded= add glucose back
–recognized by chaperone to be continually folded
- If can’t be folded properly after long time = more mannoses will be trimmed & recognized by OS-9 for degradation
-cant be correctly folded forever
Formation of disulfide bond:
protein responsible…
found in…
Disulfide bonds formed by PDI stabilize folded protein structure
-PDI – protein disulfide isomerase
Disulfide bonds - found in luminal protein
-Chemical bond
1st phase:
Formation of disulfide bond
- formation PDI substrate protein intermediate
- PDI and substrate is linked by disulfide bound. - e- transport transported to PDI.
-oxidizing PDI is reduced. - Oxidized Ero1 oxidizes reduced PDI
2nd phase:
Rearrangement of disulfide bond by
electron transport
transfer the electrons to substrate and the electrons eventually transformed back = will be reduced again.
Reduced PDI interact with Protein with incorrect disulfide bonds
Folding of hemagglutinin in ER aided by BiP, glycosylation & PDI
1a. BiPs bind to nascent chain to prevent non-specific aggregation
-7x N-linked oligosaccharide added
1b. -linked oligosaccharides recognized by Calreticulin & Calnexin
-So the polypeptide gains time for folding
- PDI catalyzes formation of 6x disulfide bonds to stabilize folded structure
Insertions/translocation of membrane proteins into lumen of ER:
Topology of membrane proteins
Type I – N(luminal), C (cytosolic)
-Has cleaved signal sequence
Type II – N(cytosolic), C(luminal)
Type III – N(luminal), C (cytosolic)
Type IV – N(luminal), C (cytosolic)
-More than 1 TMD = channel within ER
TMD: transmembrane domain
All TMD in membrane proteins form…
more about structure…
Forms ⍺-helix in lipid bilayer
Actual sequence TMD can be very different, but each TMD is 18-22aa
Majority of aa = hydrophobic
Insertion of type I membrane proteins into ER membrane: step by step
- ER signal sequence opens Sec61= translation & translocation of polypeptide starts
- Signal sequence cleaved by signal peptidase & degraded
- Translocation stops when TMD is met with Sec61
-TMD = internal stop-transfer anchor sequence - Translation continues in cytosol
- TMD moves laterally to Sec61(through the seam) & eventually ER lipid bilayer
- When translation completed = ribosome released into cytosol
-Leaves protein anchored in membrane
Sec61 opens transiently allow
allow TMD to exit laterally
-Like a clam
-Lateral exit seam in Sec61
–Between space between ⍺-helix
