L17: Translocation Of Proteins Flashcards
Rough ER
Ribosomes attached
Protein import into ER
Smooth ER
No ribosomes attached
Typically tubule network
Transport ER products to various destinations
Eukaryotic cotranslation translocation
Synthesis of secretory & membrane proteins is coupled to translocation across ER membrane
Features: signal recognition particles, signal receptors, translocons (participate in secretory protein translocation), signal peptidase, energy requirement- GTP
Cotranslational translocation
- Initiation of protein synthesis. Signal sequence synthesised first
- Signal sequence bound by signal recognition particle (SRP)
- SRP binds to SRP receptor. Interaction strengthened by binding of GTP
- Opening of translocon and insertion of signal sequence. Hydrolysis of GTP and dissociation of SRP
- Signal sequence cleaved by signal peptidase
- Elongation
- Release of ribosome and closing of translocon
How are membrane proteins synthesised with right orientation?
Integral membrane proteins
Synthesised rough ER
Embedded in ER membrane in their unique orientation
Transport to location maintains orientation
ER proteins, golgi proteins, plasma membrane proteins & lysosomal proteins
Orientation is established during biosynthesis on ER membrane
Protein orientation: Type II proteins
Lacks cleavable N-terminal ER signal sequence
Signal anchor sequence: functions as both ER signal sequence and membrane anchor sequence
Synthesis of cytosolic ribosome, synthesis of signal-anchor sequence, bound by SRP
- Binding to ER membrane
- Internal signal-anchor moves out of translocon and peptide chain extruded into ER lumen
- C-terminus released into ER lumen & ribosomal subunits released
Protein orientation: type III proteins
Lacks cleavable N-terminal ER signal sequence
Signal anchor sequence: functions as both ER signal sequence and membrane anchor sequence
Synthesis of cytosolic ribosome, synthesis of signal-anchor sequence, bound by SRP
- Binding to ER membrane
- Internal signal-anchor moves out of translocon and peptide chain extruded into cytosol
- C-terminus released into cytosol & ribosomal subunits released
Protein orientation: type I proteins
Stop transfer sequence: hydrophobic sequence that becomes membrane-spanning alpha helix
- Hydrophobic sequence synthesised and prevents (stops) further extrusion into ER lumen
- Lateral movement (transfer) between translocon and lipid bilayer
5-6. Synthesis continues with peptide extruded into cytosol
Translocation
Proteins delivered to proper cell compartment by translocation (cotranslational & post-translational)
Signal sequence of targeting sequence
Directs protein to its appropriate location in cell
Mitochondria
Features:
Most of ATP generated in cell
Have own protein synthesising machinery but only makes small no. of proteins
Most mitochondrial proteins are encoded by nuclear genes
Most mitochondrial proteins synthesised on cytosolic ribosomes
How do mitochondrial proteins gain access fo proper subcompartment?
All info required to target precursor protein from cytosol to mitochondria matrix is contained within its N-terminal uptake targeting sequence
Requires energy
Translocation occurs at point where outer and inner organelle membranes are in close contact - contact site
Sorting proteins to correction location requires:
Sequential targeting sequences on protein
Proteins sorted to destinations other than matrix usually contain 2 or more targeting sequence
Sequential translocation systems
TOM: translocon of outer membrane. All imported proteins interact with TOM complex
TIM: translocon of inner membrane
Matrix targeting sequences
Usually N-terminus
20-50 AA long
Rich in hydrophobic and +vely charged AA
Targeting sequence
Adopts helical conformation
Structure of amphipathic alpha helix having basic (+) residues on one side and uncharged and hydrophobic residues on other
Mitochondrial presequences contain positively charged amphipathic alpha helices
Post translational translocation- mitochondria. Example: protein import into mitochondrial matrix
- Only unfolded proteins can be imported into organelles. Cytosolic chaperones (Hsc70) maintain precursors of mitochondrial proteins in an unfolded state.
ATP required - Bind to import receptor (Tom 20/22) on outer mitochondrial membrane
- Transfer to general import pore (Tom40)
- Translocation through outer membrane (Tom40)
- Translocation through inner membrane (Tim44). Energy from proton-motive force across inner membrane and ATP hydrolysis by Hsc70 ATPase helps drive import
- Uptake-targeting sequence is removed by matrix protease
- Folding into mature, active formation
Translocation of proteins
Proteins delivered to proper cell compartment by translocation
Cotranslational translocation: synthesis of polypeptide. Contains signal sequence -> cause ribosomal mRNA complex to bind to ER. Protein synthesised in ER or as transmembrane protein
Post-translational translocation: ribosomes synthesise proteins (synthesised in cytosol). Proteins have targeting sequence -> allow proteins to find right location
Cleavable signal sequence features
No sequence consensus; structural properties important
One or more positively charged residues (arginine, lysine, histidine)
Hydrophobic residues (nonpolar)
Cotranslational translocation features
Amino-terminal signal sequence
10-15 hydrophobic AA residues
One or more positively charged residues (usually near amino terminus, usually precedes hydrophobic sequence)
Cleavage site- relatively polar or short side chains near cleavage site
Type 1 features
Signal sequence cleaved off
N-terminal in ER
Internal stop-transfer anchor sequence: transmembrane domain
Type 2 features
Synthesis of N terminal in cytosol
Synthesis of rest of polypeptide in lumen of ER
Internal signal anchor sequence: transmembrane domain
No cleaved signal sequence
Type 3 features
No signal sequence cleaved off
Internal signal-anchor sequence: transmembrane domain
Rest synthesised in cytosol
