Transporting proteins into Membranes Flashcards
Signal Based targeting
Major Protein sorting pathway:
- targets newly synthesised proteins from cytosol to an organelle (during translation or following synthesis)
- Soluble proteins are translocated across membrane into aqueous organelle interior
The Rough Endoplasmic Reticulum (RER) in protein synthesis and trafficking
ONLY proteins destined for the cytosol, nucleus, mitochondria or peroxisomes DON’T enter the RER:
- most other proteins are cotranslationally translocated into the RER (otherwise they undergo post-translational translocation: enter the RER following translation by cytosolic ribosomes)
Targeting proteins to the RER
Soluble proteins: mRNA’s N-terminal sequence directs protein/ribosome complex to the ER for co-translational translocation to occur.
- N-terminal sequence: hydrophobic, variable, 16-30 residues long (6-12 hydrophobic amino acids), attracted to ER membrane
Experimental evidence of simultaneous translation and translocation using microsomes (and Pulse Chase)
Producing microsomes in vitro from homogenized eukaryotic cells.
Those with ribosomes bound were isolated using differential and sucrose density-gradient centrifugation
Pulse chase can be carried out using these
Pulse Chase
Tracking radiolabelled proteins inside the RER:
1) incubate cells in radiolabelled amino acids (newly synthesised = labelled)
2) Microsomes treated with protease to remove any protein NOT protected by the ER (radiolabelled ones are safe)
3) Add detergent to dissolve the ER membrane, no longer protecting the newly synthesised proteins - they get lysed - fluorescence observed
Proof of newly synthesised entering RER
Proteins invloved in initialising co-translational translocation
1) Signal Sequence Recognition protein
- mechanism that targets secretory protein to ER
2) Signal Recognition Particle (SRP) and its receptor
- Bind to ER signal of nascent protein and large ribosomal subunit
- SRP = 6 proteins bound to 300nt RNA (ribonucleoprotein)
- Its p54 subunit binds the hydrophobic residues of Signal Sequence
- SRPR binds SRP: release upon GTP hydrolysis
Translocon
Binds signal sequence once it’s released by the SRP and facilitates the insertion of polypeptide into ER membrane
- forms channel through which the polypeptide is passed
- formed from Sec (secretory) proteins with a central translocation channel and various other components including a signal peptidase to cleave the SRP
Signal Peptidase
A serine protease in ER lumen, associated with translocon
- active site endoproteolytically cleaves signal sequence located at the extracytoplasmic site of the membrane
Unidirectional Movement across ER
Sec63/BiP interaction
Protein chaperones (Sec63 complex/BiP) bind to and stabilise the unfolded protein - The Heat Shock Protein (HSP) BiP contains a peptide binding domain and an ATPase domain
1) Sec63 hydrolyses BiP: ATP promotes conformational change
2) BiP-ADP binds to the protein as translocation continues, preventing the protein from ‘sliding back’ and stabilising it
3) BiP releases upon maturation and binds to ATP - the cycle repeats
Oligosaccharyltransferase (OST)
Catalyses N-linked glycosylation during co-translation
- uses energy from the cleavage of pyrophosphate bond between the dolichol-glycan molecule
- attaches glycan to nitrogen atom (aspargine) of protein: attachment requires consensus sequence (Asn-X-Ser/Thr)
Membrane Protein classes
Type 1 (e.g. LDL receptor)
- Cleaved N-terminal ER sequence
- N-terminal = lumenal; C-terminal = cytosolic
Type 2 (e.g. Transferin receptor)
- No cleavable N-terminus
- N-terminal = cytosolic; C-terminal = luminal
Type 3 (e.g. Cytochrome 450)
- Same orientation as Type 1 but no cleavable N-terminus
- Different position of positive charges to type 2 (it’s on the C-terminal side)
Stop-Transfer Anchor Sequence (STA)
Embedded into the membrane, following which translation continues to occur but growing into the cytosol
Type 1 - Integral Membrane Protein
Signal Peptidase cleaves Signal Sequence and translation continues
- When hydrophobic transmembrane domains (TMDs) enter translocon, translocation stops, the translocon opens laterally and the TMD integrates into ER membrane
Type 2 - Transmembrane Protein Insertion
No N-terminal Sequence: instead internal sequence in polypeptide recognised by SRP
- inserted into translocon pore, the signal acts as a TMD
- released by the translocon, it embeds into the ER bilayer (a.k.a. Signal Anchor (SA) sequence)
Type 3 membrane protein
Several transmembrane sequences (STAs and SAs)
- each time sequence enters translocon it integrates (different orientations depending on signals and sequences, hence different classified types)