Lecture 9 Flashcards
What is the signal sequence hypothesis?
Microsomes added to proteins normally secreted out of the cell –> protein is smaller b/c signal peptide gets cleaved –> lower on SDS PAGE gel than w/o microsomes
If a protein is higher on the SDS PAGE, what can be said about its signal peptide? Lower?
- Higher = protein has a signal peptide
- Lower = signal peptide has been processed/cleaved
How can you identify on the SDS PAGE a protein that is normally secreted?
Normally secreted proteins treated w/ microsomes and protease have a band the same height as when treated w/ just microsomes
List the 6 major players in the mechanism of protein translocation.
- mRNA
- Ribosome
- Signal peptide
- SRP
- SRP receptor
- Pore in the ER
What is the role of mRNA in protein translocation?
Codes for the protein to be translocated across ER membrane
What is the role of ribosomes in protein translocation?
Translates mRNA into a peptide
What is the role of the signal peptide in protein translocation?
Targets the protein/mRNA/ribosome complex to the ER or translation and translocation
What is a signal peptide composed of?
6-15 hydrophobic residues
Where are signal sequences normally found on a secreted protein? Transmembrane protein?
- Normally @ the N-term of a secreted protein
- Can be found in the middle of a transmembrane protein
What does the location of a signal sequence on a protein dictate?
Topology of the protein
What is SRP? Role in protein translocation?
Signal Recognition Particle: recognizes signal sequence, arrests translation, and targets ribosome to ER membrane
What is SRP composed of?
6 peptides and 1 RNA molecule
What is a major component of the ER pore?
Sec61
Where does all protein synthesis occur? Why?
All protein synthesis occurs in the cytoplasm b/c all ribosomes are in the cytoplasm
Describe the process of protein translocation across the ER membrane?
- SRP in cytoplasm (GDP state) binds to signal sequence on mRNA/ribosome/protein complex
- Ribosomes act as a GEF for SRP –> converts from GDP to GTP state
- SRP acts as GEF2 for SRP receptor –> converts from GDP to GTP state
- Complex binds to SRP receptor b/c both in GTP state
- Translocon (Sec61) functions as GAP –> both SRP and SR convert from GTP to GDP
- SR releases complex to translocon
- SRP is released from complex
- Plug on translocon opens –> allows unfolded peptide to enter ER lumen
You have isolated a yeast strain with a mutation in the GTP binding domain of SRP. This mutant SRP can not bind a nucleotide. Where would you predict to find a protein that is normally secreted?
In the cytoplasm
If you add nuclear localization sequence PKKKRKV to the middle of BiP protein that resides in ER lumen, where would you expect to find BiP?
ER b/c NLS overriden by signal sequence for ER since receptors will read what it on the end first
How does the pore of the translocon contribute to topology?
CHARGES: pore surrounded by charged residues on cytoplasmic surface –> protein w/ opposite charged residues will end up on the cytoplasmic side of the membrane
What are the characteristics of Type I membrane proteins?
- N-term in ER lumen
- C-term in cytosol
- Has signal sequence
- Has stop transfer sequence (STS) in the middle
- Single pass
What are the characteristics of Type II membrane proteins?
- N-term in cytosol
- C-term in ER lumen
- Internal start signal sequence (ISS)
- (+) charged AAs before ISS
- Single pass
Which topology of membrane proteins is relatively rare?
Type III
What are the characteristics of Type III membrane proteins?
- N-term in ER lumen
- C-term in cytosol
- No signal peptide
- (+) charged AAs after ISS in cytosol
- Single pass
Which 2 membrane protein topologies are similar?
Type I and III
What is the difference b/t Type I and III membrane protein topologies?
- Type I use N-term signal sequence to co-translationally translocate N-term into ER lumen
- Type III use internal signal w/ (+) charges to determine its topology and pushes N-term into the ER after translation
What are the characteristics of all Type IV membrane proteins?
- Multi pass
- May or may not have signal peptide
- Domains alternate b/t ER lumen and cytoplasm
- C-term in cytoplasm or lumen based on # of membrane passes/spans
What are the characteristics of Type IV-A membrane proteins?
- N-term in cytosol
- ISS-STS-ISS-STS
What are the characteristics of Type IV-B membrane proteins?
- N-term in ER lumen
- (+) charged AAs after 1st ISS
- ISS-ISS-STS-ISS-STS or SSS-STS-ISS-STS-ISS
What identifies the signal sequence on a hydrophobicity plot?
First peak
