Protein Translocation Flashcards
Describe transmembrane transport / protein translocation
From cytosol to mitochondria, plastids, peroxisomes, endoplasmic reticulum
can be co translational or post translational
What are the 3 options for when a protein is made?
- No targeting/signal sequence - released from ribosomes and enter cytosol (default pathway)
- Organelle specific targeting sequence - released from ribosome then imported (into mitochondria, chloroplast, peroxisomes, NLS etc)
- ER signal sequence - enter ER and join secretory pathway
Describe the signal hypothesis
Gunter Blobel - Signal Hypothesis
Discovered that proteins have intrinsic signals that govern their transport and localisation in the cell - I.e proteins have target post codes
Proteins have a signal sequence which guides the positioning of the ribosome to ER membrane and directs newly synthesised polypeptides into endoplasmic reticulum to cross membrane
What are signal peptides/patches?
Signal peptides/patches are regions of amino acids that can be contiguous (peptide) or discontiguous (patch) found on proteins.
Signal patches: distant regions in primary sequences but come closer together in tertiary fold
* cannot be cleaved
Signal Peptides: 2D continuous stretch found on N-Terminus of proteins
* Highly conserved therefore bioinformatics can be used to predict whether signals are present
* about 20 amino acids
have positive N- terminal, hydrophobic middle, polar, hydrophillic end
-> signal peptides can be manipulated to change destination of a protein
What is a signal recognition particle (SRP) ?
SRP found in cytosol. binds to signal sequence and cleft on ribosome. This stalls translocation of polypeptide until it is bound to ER
* Dual recognition of signal peptide by SRP in cytosol and by SRP receptor on RER membrane allows selective proteins in ER
What is SEC translocon?
SEC translocon is a heterotimeric complex, helical pore composed of 3 subunits: SecY, SecE, SecG
- used in both co and post translational translocation
- found in ER membrane
- evolutionary conserved from mammals to bacteria (secY)
- Passive pore: associating partners provide energy (GTP hydrolysis in co-translational translocation)
- Gated pore: closed by plug when not used
pores open laterally
- aqueous channel, hydrophobic lining
- helical plug blocks channel in closed state, polypeptide displaces the plug and is translocated, unfolded through core’s hydrophobic lining
What is co translational protein translocation?
- occurs at the same time polypeptide chain is being made
- more common
- signal sequence found only on N terminal of polypeptide chain
SEC Translocon = a passive pore, energy comes from GTP hydrolysis in co translational translocation
- In cytosol, ribosome begins to translate mRNA from 5’ end
- SRP binds to signal peptide and ribosome cleft which halts translation of mRNA to polypeptide
- SRP binds to SRP receptor on RER
- GTP binds to SRP and SRP receptor and triggers transfer of signal sequence from SRP to SEC translocon
- Hydrophilic beginning of signal sequence from polypeptide interacts with SEC translocon and plug of SEC translocon is displaced as polypeptide moves through into RER lumen
- Hydrolysis of GTP to GDP leads to SRP and SRP receptor dissociating and recycled
- Polypeptide translocates through SEC translocon pore
- Signal sequence cleaved by signal peptidase in RER lumen
- Ribosome continues to translate mature polypeptide which is released into RER lumen
- Translocator closed
What is post-translational modification?
- found in yeasts and higher eukaryotes
- signal sequence can be found anywhere on polypeptide chain
- Translocating polypeptide chain enters SEC translocon
- Signal sequence cleaved by signal peptidase
- To ensure unidirectionality and Brownian Ratchet, sec63 complex is bound to BiP which is bound to ATP
- ATP is hydrolysed to ADP
- BiP in ADP bound state binds to polypeptide chain to prevent further backflow
- does not require SRP or receptor
Describe the biosynthesis of integral membrane proteins
TYPE I - NH3+ in lumen, COO- in cytosol
- have signal sequence at N terminus and internal stop transfer sequence
1. Co translational translocation initiated by N terminal ER signal sequence
2. Stop transfer sequence stops the transfer of nascent peptide through translocon
3. Signal peptidase cleaves signal sequence
4. Stop transfer sequence interacts with binding site inside translocon causing it to change conformation and discharge the protein laterally into bilayer
TYPE 2 - NH3+ in cytosol, COO- in lumen
- have internal signal sequence
TYPE 3 - COO- in cytosol, NH3+ in lumen,
- have multiple transmembrane domains in a single polypeptide
- have internal signal sequence
TYPE 4 (multipass transmembrane protein)
- have multiple hydrophobic regions embedded in bilayer
- internal ER signal sequence acts as start transfer signal and initiates transfer of the C terminal part of the protein
- Stop transfer sequence enters translocator and discharges protein laterally into membrane
How do topogenic sequences predict orientation?
A topogenic sequence is a collective term used for a peptide sequence present at nascent proteins essential for their insertion and orienting in cellular membranes.
- usually first transmembrane helix determines orientation
- can identify transmembrane proteins
- look 15 amino acids either end: more +ve end is cytosolic
