2.1 - Transmembrane Transport (Insertion into ER Membrane) Flashcards
how is a protein inserted into the ER lumen/membrane?
- ribosome docks onto ER membrane surface - injects polypeptide in the ER lumen as it is being synthesized - ER signal sequence cleaved off by signal peptidase
Type 1 ER protein
ER protein with one TM segment and an ER signal sequence (amino end in ER lumen)
Type 1 ER protein - topology
s18
Type 1 ER protein - hydropathy plot
s19
Type 2 ER protein
ER protein with one TM segment start-transfer sequence (amino end in cytosol)
Type 2 ER protein - topology
s20
Type 2 ER protein - hydropathy plot
s21
signal-anchor sequence
found in type 2 and 3 ER proteins
- start-transfer sequence near N-term of protein that is a TMS recognized by SRP
- positive is always facing the cytosol
why is the positive charge of the start-transfer sequence of type 2/3 ER proteins always towards the cytosol?
negatively charged lipids are present around the translocation channel
Type 3 ER
ER protein with one TM segment start-transfer sequence (amino end in ER lumen)
Type 3 ER protein - topology
s23
Type 3 ER protein - hydropathy plot -
probably same as type 2
Type 4 ER protein
multi-pass ER protein with a start transfer-sequence and a start-transfer sequence (can have multiple
positive-inside rule with alkaline phosphatase
1) hairpin loop inside = blue
2) hairpin loop outside = white
explain the charge gradient across the ER membrane at the translocon
- cytoplasmic side of ER membrane (-)/basic
- ER lumen side of ER membrane (+)/acidic
- makes a charge gradient across the membrane, so harder to insert + residues across the membrane
- expect that cytosolic loops will have more + charged residues
SecYEG
translocation channel/translocon of prokaryotes
Sec61aBg
translocation channel/translocon of eukaryotes
SecYEg
translocation channel/translocon of archaea
translocon
membrane channel in the ER membrane through which the polypeptide chain is transferred through
translocon structure
membrane channel protein made of 3 subunits
- pore ring made of 6 isoleucines gated by a short helix when closed
- SecY/Sec61 alpha
how can protein structure be determined experimentally?
X-ray diffraction of protein crystals
- beam of X-ray (short wavelengths) directed across crystal
- some X-rays are scattered in waves based on the structure and the diffraction pattern can be used to make an electron density map
SecYEG
Sec61aBg
???
what happens if you remove the plug from translocon
cell dies
- membrane loses impermeability to ions
what is the translocon pore ring made of
6 isoleucines (hydrophobic amino acid resides)
what is the function of the pore ring
forms a gasket around the polypeptide chain in transit so that it is ion tight
what is the equivalent of the Sec61B subunit in bacterial translocon?
SecG
what is the equivalent of the Sec61g subunit in bacterial translocon?
SecE
what is the purpose of the seam opening of Sec complex
lateral opening along seam allows the translocating peptide chain to access the lipid bilayer
how is the TMS released from the Sec complex
two halves of SecY/Sec61a opening
describe secondary protein structure of porins
beta-barrel proteins
why are porins not found in the IM of bacteria
???
two modes of protein translocation (2)
1) co-translational translocation
2) post-translational translocation
co-translational translocation
translocation during translation
post-translational translocation
translation before translocation. Chaperone proteins maintains the proteins in a loosely folded state
how does post-translational translocation work in bacteria?
SecA ATPase uses ATP to push the protein across the SecY complex (ratchet mech)
how does post-translational translocation work in yeast?
BiP ATPase binds polypeptide chains as it emerges from the pore (pulling mechanism)
what kind of translocation occurs across the ER membrane
co-translational translocation
what does the translocator need to feed the polypeptide through the pore
accessory proteins (SecA ATPase in bact, BiP ATPase in euk)
is yeast prokaryotic or eukaryotic
eukaryotic (has a nucleus)
BiP ATPase function (2)
- protein that pull proteins in post-translational translocation (yeast) across ER membrane
- also acts as a chaperon
OST complex location
oligosaccharide transferase complex
- located in ER lumen
OST complex function
glycosylate proteins that are in the ER lumen
what proportion of eukaryotic proteins are glycosylated
50%
why are so few proteins in the cytosol are glycosylated
OST (oligosaccharide transferase) are only located in the ER lumen
how does the carbohydrate layer of the ER useful for the cell
protects cells against mechanical and chemical damage
how does glycosylation in ER lumen work
N-linked glycosylation - oligosaccharide transferred to Asn on polypeptide via oligosaccharide transferase
what holds the preformed oligosaccharide in the ER lumen prior to it being used for glycosylation
dolichol (special lipid) holds preformed oligosaccharide (14 sugar units) in ER lumen via a high energy pyrophosphate bond
where does O-linked glycosylation occur and what proportion is it performed in
In the Golgi
what is O-linked glycosylation
preformed oligosaccharide linked to OH group of serine (S) or threonine (T)
what are oligosaccharides used for
used as tags to mark the state of protein folding
calnexin
ER chaperone protein
- binds to oligosaccharide on incompletely folded proteins and retains them in the ER until the protein is folded
calreticulin
ER chaperone protein - binds to oligosaccharide on incompletely folded proteins and retains them in the ER until the protein is folded
lectin
carbohydrate-binding proteins
PDI
protein disulfide isomerase
- protein in the ER that helps the formation of S-S bonds
where do disulfide bonds form
in the ER usually, the reducing environment makes it hard for S-S bonds to form in the cytosol
GPI
glycophosphatidylinositol anchor
GPI function
1) protein transportation
2) cell adhesion
3) cell wall synthesis
4) cell surface protection
how is GPI added onto proteins?
ER protein, GPI-transamidase, cleaves off C-terminal transmembrane segment and attaches GPI anchor to C-terminus of protein
what kind of proteins is GPI found on
plasma membrane proteins
HA
influenza hemagglutinin - antigenic glycoprotein responsible for binding of virus to cell (target is sialic acid)
why is trypanosoma brucei so potent
parasite that is able to cross blood brain barrier
- VSG (variable surface glycoprotein) coat prevents the immune system from accessing the PM epitopes of the parasite
- allows it to evade the immune system
- antigenic variation allows it to avoid specific immune responses
