L17: Translocation Of Proteins

Question 1

Rough ER

Answer 1

Ribosomes attached

Protein import into ER

Question 2

Smooth ER

Answer 2

No ribosomes attached

Typically tubule network

Transport ER products to various destinations

Question 3

Eukaryotic cotranslation translocation

Answer 3

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

Question 4

Cotranslational translocation

Answer 4

1. Initiation of protein synthesis. Signal sequence synthesised first
2. Signal sequence bound by signal recognition particle (SRP)
3. SRP binds to SRP receptor. Interaction strengthened by binding of GTP
4. Opening of translocon and insertion of signal sequence. Hydrolysis of GTP and dissociation of SRP
5. Signal sequence cleaved by signal peptidase
6. Elongation
7. Release of ribosome and closing of translocon

Question 5

How are membrane proteins synthesised with right orientation?

Answer 5

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

Question 6

Protein orientation: Type II proteins

Answer 6

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

1. Binding to ER membrane
2. Internal signal-anchor moves out of translocon and peptide chain extruded into ER lumen
3. C-terminus released into ER lumen & ribosomal subunits released

Question 7

Protein orientation: type III proteins

Answer 7

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

1. Binding to ER membrane
2. Internal signal-anchor moves out of translocon and peptide chain extruded into cytosol
3. C-terminus released into cytosol & ribosomal subunits released

Question 8

Protein orientation: type I proteins

Answer 8

Stop transfer sequence: hydrophobic sequence that becomes membrane-spanning alpha helix

3. Hydrophobic sequence synthesised and prevents (stops) further extrusion into ER lumen
4. Lateral movement (transfer) between translocon and lipid bilayer

5-6. Synthesis continues with peptide extruded into cytosol

Question 9

Translocation

Answer 9

Proteins delivered to proper cell compartment by translocation (cotranslational & post-translational)

Question 10

Signal sequence of targeting sequence

Answer 10

Directs protein to its appropriate location in cell

Question 11

Mitochondria

Answer 11

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

Question 12

How do mitochondrial proteins gain access fo proper subcompartment?

Answer 12

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

Question 13

Sorting proteins to correction location requires:

Answer 13

Sequential targeting sequences on protein

Proteins sorted to destinations other than matrix usually contain 2 or more targeting sequence

Question 14

Sequential translocation systems

Answer 14

TOM: translocon of outer membrane. All imported proteins interact with TOM complex

TIM: translocon of inner membrane

Question 15

Matrix targeting sequences

Answer 15

Usually N-terminus

20-50 AA long

Rich in hydrophobic and +vely charged AA

Question 16

Targeting sequence

Answer 16

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

Question 17

Post translational translocation- mitochondria. Example: protein import into mitochondrial matrix

Answer 17

1. Only unfolded proteins can be imported into organelles. Cytosolic chaperones (Hsc70) maintain precursors of mitochondrial proteins in an unfolded state.
   ATP required
2. Bind to import receptor (Tom 20/22) on outer mitochondrial membrane
3. Transfer to general import pore (Tom40)
4. Translocation through outer membrane (Tom40)
5. Translocation through inner membrane (Tim44). Energy from proton-motive force across inner membrane and ATP hydrolysis by Hsc70 ATPase helps drive import
6. Uptake-targeting sequence is removed by matrix protease
7. Folding into mature, active formation

Question 18

Translocation of proteins

Answer 18

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

Question 19

Cleavable signal sequence features

Answer 19

No sequence consensus; structural properties important

One or more positively charged residues (arginine, lysine, histidine)

Hydrophobic residues (nonpolar)

Question 20

Cotranslational translocation features

Answer 20

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

Question 21

Type 1 features

Answer 21

Signal sequence cleaved off

N-terminal in ER

Internal stop-transfer anchor sequence: transmembrane domain

Question 22

Type 2 features

Answer 22

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

Question 23

Type 3 features

Answer 23

No signal sequence cleaved off

Internal signal-anchor sequence: transmembrane domain

Rest synthesised in cytosol