Lecture 10 Flashcards
what transport method do proteins that are sorted to the ER use?
what other transport system can they further be sorted by?
- Proteins are sorted to the ER by transmembrane transport
➢They can be further sorted by vesicular transport to other compartments or to the cell surface (proteins that are sorted to the ER matrix space stay there vs sorted to the ER lumen stay there – look at slide 3 for diagram)
what are the major functions of the ER (2)
which proteins have an ER signal sequence?
- Major functions of the ER:
- synthesis and modifications of proteins
- synthesis of lipids
Proteins sorted to the ER have an ER signal sequence
These include:
* soluble proteins, transmembrane proteins
* proteins destined for Golgi, secretion, lysosomes
what is the common pool of ribosomal subunits in the cytosol?
- when a ribosome partakes in creating a protein, it is always recycled afterwards
- for instance a ribosome making an mRNA cytosolic protein will go through typical translation where the mRNA strand is fed through and the translation is made, and then after it reaches the stop codon of the mRNA, the ribosome dissociates and gathers in the common pool of ribosomal subunits in the cytosol
- same way, when making an ER protein, after it is co-translated, it will dissociate to the common pool
how is protein sorted in the ER (4)
- ribosome is recruited via common pool of ribosomal subunit
- translation starts
- ribosome takes the mRNA strand and translates it. the first codons it creates is the ER signal sequence (hydrophobic amino acids at the N-term)
- the ER signal sequence when bound to SRP signals for translation to pause and continue when the ribosome is translocated to the ER membrane (SRP binds to receptor).
- the ribosome is moved to the ER membrane by SRP
- translation occurs while the protein is translocated (via translocator) into the ER lumen – thus co-translational translocation
what are SRPs and how do SRPs work?
- SRPs = signal recognition particle
- it has an SRP receptor that has GTPase domains that bind GTP (not impt)
how?
- SRPs bind to the ER signal on the ribosome with high affinity.
- this causes SRP to pause translation and only resume when ribosome is translocated to the ER membrane where the SRP binds to the SRP receptor
(note: the other end of the SRP binds to the ribosome with low affinity – only the ER binding region is high affinity)
- then the ribosome forms a tight seal with the protein translocator next to the SRP receptor on the ER membrane
(note: tight seal prevents diffusions of ions, small molecules)
- after tight seal is made and the translation resumes through the translocator pore, the SRP - SRP receptor is dissociated and recycled – also conducts GTP hydrolysis
how is the ER signal sequence cleaved? where does the ER signal and protein translocator go after this and after the protein is fully synthesized?
- ER signal sequence = N-terminal start-transfer sequence
- when the ribosome is tightly bound to the protein translocator, the ER signal enter firsts and also tightly binds to the translocator
- a signal peptidase enzyme cleaves the ER signal once the protein is fully synthesized and its not needed anymore
- the ER signal sequence laterally diffuses into the lipid bilayer
- the translocated protein is released into the ER
- the translocator is reused
what are single and multipass transmembrane proteins?
how many single pass transmembrane protein insertions are there?
- single pass transmembrane proteins pass through the ER membrane once
- multipass- pass through membrane multiple times
there are three types of single pass TM proteins:
- 1: ER start signal is cleaved, and TM protein is the stop codon (like an integral protein)
- 2: start signal is TM, N term adjacent AA is pos in cytosol, C term adjacent AA is neg in ER lumen.
- 3: start signal is TM, N term adjacent AA is neg in ER lumen, C term adjacent AA is pos on cytosol.
describe single pass transmembrane protein 1 as denoted in notes:
- cut the NH2
- ER signal sequence: (NH2) start-transfer
- translocator binds to the N terminal start transfer ER sequence
- as you make the rest of the protein into the cytosol with the ribosome, the mRNA codes for the stop transfer sequence in the middle since thats how the mRNA codes the protein
- the signal peptidase cuts the start signal peptidase that is bound to the translocator and that diffuses latterly as does the translocator protein
- as this mRNA strand enters the membrane, the stop codon which was made in the middle stops protein from transporting fully into the ER lumen. thus making a TRANSmembrane protein
- the N terminal is in the ER and the C terminus is in the cytosol.
describe single pass transmembrane protein 2&3 as denoted in notes:
- internal start transfer sequence
- the ribosome codes for the TM domain as an INTERNAL start transfer sequence (not at the beginning like the first ex.)
NOTE: the more positive side of the amino acids adjacent to the INTERNAL start transfer sequence will stay in the cytosol. the more negative will enter the ER lumen
cytosol = +
ER = -
- the mRNA feeds through the translocator, with the INTERNAL start transfer sequence bound to the translocator on the membrane – both the C and N terminus are in the cytosol (loop)
- the difference between single pass 2 and 3, is the orientation at which the mRNA feeds through the translocator.
- In single pass 2 = N term –> start signal –> C term
- in single pass 3 = C term –> start signal –> N term
- after the loop is made with the N and C term on the cytosolic side, the translocator is dissociated
- this causes the more negative AA adjacent region to enter the ER and the more pos to stay at the cytosolic side
–> the C end in the ER (-) and N end in the cytosol (+) – the INTERNAL start domain is docked on the membrane.
- look at diagram slide 11
explain how multipass transmembrane proteins are made via 1st TM internal start sequence followed by 2nd TM stop sequence
- as in protein sorting 2&3 the internal start sequence is coded and docked on the translocator with the N and C term facing the cytosol
- however, later on as the ribosome is still translating the protein, it codes for a stop codon
- this stop codon then also gets docked on the ER membrane
- thus the N and C term face the cytosol with a loop in the ER membrane (2 pass TM protein)
- the N and C term face the cytosol due to the adjacent AA next to the internal start codon. they happen to be positive towards the N terminus.
Look at diagram slide 12
explain multipass TM proteins with rhodopsin as an example
lowkey dont understand
- 1st TM → start-transfer; (+) amino acids, cytosolic
- 2nd TM → start-transfer
- 3rd TM → stop-transfer
- 4th TM → start-transfer
- The N terminus is negative, it will be on the ER lumen side, and the C terminus will be on the cytosol side
- to become multi passed, the next TM will be a start codon as well so it can get passed through again?
what is a GPI anchored protein and how is it formed
- a protein that is fully on one side (not integral)
- lipid phosphate bound to TM on one side and protein bound on other side.
how is it formed?
- starting product: TM stop codon with C terminal on cytosol side (+) and N term on ER side (-)
- this starting product is a signal to detect and call over the GPI anchor.
- an ER enzyme transfers the protein on the ER lumen side to the GPI anchor
- the cleaved C-terminal peptide degrades, and the protein is now bound to the GPI anchor
- in the ER it will end up being in the luminal side and can go to the exterior surface
