Lecture 7 - endomembrane system part 2 Flashcards
What are the three pathways in the endomembrane system?
biosynthetic, secretory, endocytic pathways
What is the starting point for both secretory and biosynthetic pathways?
ER
What is ER the site of?
protein and lipid synthesis, protein folding, and processing quality control
What is the ER?
highly complex network of membrane enclosed, rod like tubules and sheet like cisternae, one of two main sites in cell for protein synth (translation)
what organelle has the largest SA?
ER
What is the ER lumen?
aqueous space inside ER tubules and cisternae
What mediates tubules and cisternae in the ER?
reticulons
What do ER integral membrane proteins possess?
hair pin secondary structure, which regulates ER membrane curvature and the overall shape of the ER
ER cisternae vs tubule?
cisternae is a flattened pancake like structure and tubule is like a tube
Is the ER a dynamic network?
yes, ER tubules and cisternae in constant flux, growing bending fusion fission etc.
What is the difference between RER and SER?
RER - mostly cisternae with bound ribosomes, involved in protein and membrane phospholipid synthesis
SER - mostly curved tubules lacking ribosomes, involved in Ca storage, and hormone synthesis
How many ER subdomains are there?
> 20
- nuclear envelope
mitochondra and plasma membrane associated membranes
- ER exit sites - ER regions where transport vesicles bud off en route to golgi
What are the two types of ribosomes?
- free ribosomes in cytoplasm
- fate of nascent properly folded soluble or membrane protein in cytoplasm - ER membrane-bound ribosomes
- fate of nascent, properly folded soluble or membrane protein in RER
What are the steps to cotranslational translocation of soluble protein into the RER lumen?
- in the cytoplasm, tln of mRNA on free ribosome begins. N-term of nascent, growing polypeptide emerges from ribosome. N-term contains signal sequence (8-15 AA - RER targeting signal)
- exposed signal sequence recognized by signal recognition particle (SRP)
SRP binds to ribosome and stops protein tln - SRP targets complex to surface of RER
SRP binds to SRP receptor
cytoplasmic facing domains fo SRP receptor serve as docking site for incoming SRP
interaction between SRP and SRP receptor strengthened by both binding GTP - GTP hydrolysis results in release of SRP and SRP receptor (used for additional rounds of import)
simultaneously, nascent polypeptide and ribosome transferred to cytoplasmic side of Sec61 translocon
transfer of nascent polypeptide ribosome to sec61 translocon results in N-terminus of nascent polypeptide inserted into opening of translocon channel
translation resumes and elongating polypeptide chain continues to pass through translocon channel towards ER lumen (driven by translation) - N-term signal sequence enters ER lumen cleavecd by signal peptidase and degraded
- co-translational translocation of polypeptide into ER lumen continues
- translation completed and ribosome released from translocon, remainder of nascent protein enters ER lumen
translocon closes - pore plug moves back in aqueous channel - nascent protein glycosylated (addition of sugars to polypeptide) and properly folded by reticuloplasmins
What is a SRP?
ribonucleoprotein particle consisting of 6 proteins and 1 small RNA
What is an SRP receptor?
hetero dimeric ER integral membrane protein complex
What is sec61 translocon?
multi-protein complex, consists of several ER integral membrane protein subunits forming hourglass-shaped aqueous channel
What does the hourglass shaped translocon channel of sec61 contain?
pore ring, ring of 6 hydrophobic AA located at the narrowest diameter of the channel, serves as a gate to seal channel to ions/small molecules (including during translocation) - helps maintain ER compartmentalization
What else blocks the translocon channel?
alpha helix plug
second gate-keeping mechanism (first is pore ring) during protein translocation growing polypeptide forces plug away from channel
What is signal peptidase?
ER integral membrane protein (protease) associated with translocon - catalytic domain of signal peptidase faces Er lumen. peptidase recognizes cleavage sequence motif at C-terminal end downstream of signal sequence
What are reticuloplasmins?
include BiP, calnexin and calreticulin
ER molecular chaperones - bind to nascent proteins and mediate proper protein folding (prevent protein aggregation) and oligomeric assembly
where are membrane proteins synthesized?
membrane bound ribosomes at RER, only exception are membrane proteins destined for mitochondria or chloroplasts
How is ER membrane protein insertion similar in integral membrane proteins in comparison to soluble proteins for co translational translocation?
similar initially, growing polypeptide recognized by SRP; SRP delivers stalled nascent polypeptide ribosome complex to SRP receptor; nascent polypeptide ribosome transferred to sec 61 translocon; SRP/SRP receptor released; co-translational translocation continues
EXCEPT, important mechanistic differences resulting in mature membrane protein being integrated (anchored) in ER membrane and with proper topology
What is a transmembrane domain? (TMD)
typically alpha helix of 16-25 hydrophobic AA (energetically favourable within hydrophobic interior of phospholipid bilayer)
What is membrane protein topology?
number of membrane spanning domains and orientation
What are the different classes of integral membrane proteins synthesized at the ER?
Type 1 - 1 TMD, N in-C out, signal sequence
Type 2 - 1 TMD, N out - C in, no signal sequence, SA
Type 3 - 1 TMD, N in-C out, no signal sequence, SA
tail-anchored protein - 1 TMD, N out - C in, c-term TMD
Type 4 - >2TMDs, varied topologies, no signal sequence, SA and STA
GPI anchored protein - no TMDs, N in- C in, c-term phospholipid anchor sequence
What are the steps to Cotnl tslctn for type I membrane protein?
N ER lumen - C cytosol
1. nascent polypeptide-ribosome complex targets to and associates with translocon (similar to ER lumenal protein targeting)
- N-term of nascent polypeptide enters ER lumen and signal sequence cleaved by signal peptidase
2. polypeptide co-translational translocation cont.
3. eventually first and only hydrophobic TMD enters translocon
- TMD serves as stop transfer anchor sequence (STA)
5. STA moves laterally out of translocon and becomes anchored in adjacent phospholipid lipid bilayer. tsln continues, elongating polypeptide end extends into cytosol
6. translation completed ribosome released. nascent protein diffuses away laterally in ER membrane bilayer
final membrane orientation: Ner lumen - C cytosol
What is STA sequence?
stops further translocation of polypeptide through translocon
What are the steps to Cotnl tslctn for type II membrane protein?
N cytosol- C er lumen
1. proteins signal anchor (SA) sequence enters translocon. SA flipped in translocon so N-term of polypeptide faces cytosol
2.translation continues, elongating polypeptide extends into ER lumen via translocon.
also SA moves laterally out of translocon and becomes anchored in adjacent membrane bilayer
3. translation completed, ribosome released, nascent protein diffuses away laterally in ER membrane bilayer
final membrane orientation:
N cytosol - C er lumen
What are some characteristics of Cotnl tslctn for type II membrane protein?
N cytosol- C er lumen
- opposite orientation to type I (no n-term signal sequence), possess SA sequence
- first and only TMD functions both as signal sequence for binding SRP and mediating nascent polypeptide ribosome complex targeting to translocon and as membrane anchor
What is the orientation of SA sequence in type II membrane proteins mediated by?
several +ve charged AA residues located upstream of SA, positive outside rule
outside = cytosol
inside = er lumen
What are the steps to Cotnl tslctn for type III membrane protein?
N er lumen - C cytosol
1. SRP dependent targeting to and insertion into translocon similar to type II membrane protein, but +ve charge AA located downstream c-term of SA
(SA not flipped)
2. translation continues, elongating polypeptide extends into cytoplasm
3. translation completed, ribosome released, protein diffuses away laterally in ER membrane bilayer
final membrane orientation:
N er lumen - C cytosol
What are the characteristics to Cotnl tslctn for type III membrane protein?
- same orientation as type I, but like type II, possess internal SA sequence
What are some characteristics of type IV er membrane proteins?
- multiple TMDS lack an N term signal sequence
- contain both internal SA sequences (serve to target protein to ER in SRP-dependent manner and anchor protein in ER membrane depending on positive outside rule) and internal STA sequences (serve to stop transfer of protein through and anchor into ER membrane)
Where do free ribosomes in cytoplasm go? (two fates)
remain in cytoplasm or
targets (post-translationally to proper intracellular compartment)
Where do ER membrane bound ribosomes go? (three fates)
remains in RER or localizes (moves laterally in ER membrane or lumen) to another ER subdomain or
localizes to other Er derived organelles or
targets (via transport vesicles) from ER onto another (post-ER) compartment in endomembrane system
