Transcript Signal Hypothesis - Bio 5068
The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Tuesday 9/2 2014 Mike Mueckler [email protected]
Figure 6-63
Molecular Biology of the Cell
(© Garland Science 2008)
Ribosome Structure
Formation of Polyribosomes
Figure 6-76
Molecular Biology of the Cell
(© Garland Science 2008)
Intracellular Targeting of Nascent Polypeptides
• Default targeting occurs to the cytoplasm • All other destinations require a targeting sequence • Major sorting step occurs at the level of free versus membrane-bound polysomes
Figure 12-36c
Molecular Biology of the Cell
(© Garland Science 2008)
Figure 12-41a
Molecular Biology of the Cell
(© Garland Science 2008)
Ribosomal Subunits are Shared Between Free and Membrane-Bound Polysomes
Targeting information resides in the Nascent polypeptide chain
Signal-Mediated Targeting to the RER
Properties of Secretory Signal Sequences
cleavage 8-12 Residues
N ++
Hydrophobic Core
Mature Protein
15-30 Residues • Located at N-terminus • 15-30 Residues in length • Hydrophobic core of 8-12 residues • Often basic residues at N-terminus (Arg, Lys) • No sequence similarity
In Vitro Translation/Translocation System
• • • • • • • mRNA Rough microsomes Ribosomes tRNAs Soluble translation factors Reticulocyte or wheat germ lysate Low MW components Energy (ATP, creatine-P, creatine kinase)
Isolation of Rough Microsomes by Density Gradient Centrifugation Figure 12-37b
Molecular Biology of the Cell
(© Garland Science 2008)
In Vitro Translation/Translocation System
mRNA + Translation Components + Amino acid* Protein* SDS PAGE
In Vitro Translation of Prolactin mRNA
Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary MW (kd)
SDS Gel
1 2 3 4 5 6 7 8 25 22 18
Lanes:
1. Purified prolactin 2. No RM 3. RM 4. No RM /digest with Protease 5. RM /digest with Protease 6. RM /detergent treat and add Protease 7. Prolactin mRNA minus SS + RM /digest with Protease 8. SS-globin mRNA + RM /digest with Protease
Identification of a Soluble RER Targeting Factor
RM + 0.5 M KCl Centrifuge Supernate = KCl wash Pellet = KRM MW (kd) 25 22 18 8 1 2 3 4 5
Lanes:
1. No additions 2. KRM 3. KRM / digest with Protease 4. KRM + KCl wash 5. KRM + KCl wash / digest with Protease
Purification of the Signal Recognition Particle (SRP)
MW (kd) 25 22 18 8 Hydrophobic Chromatography KCl Wash SRP 1 2 3 4 5
Lanes:
1. No additions 2. KRM 3. KRM /digest with Protease 4. KRM + KCl wash /digest with Protease 5. KRM + SRP /digest with Protease
Subcellular Distribution of the Signal Recognition Particle (SRP) Where is SRP located within the cell?
47% ribosomes + polyribosomes 15% cytoplasm 38% rough endoplasmic reticulum Conclusions: • SRP likely moves between different subcellular compartments • SRP is a soluble particle that can associate with membranes and is not a permanent membrane-bound RER receptor
Structure of the Signal Recognition Particle (7SL RNA)
Figure 12-39a
Molecular Biology of the Cell
(© Garland Science 2008)
Interactions Between SRP and the Signal Sequence and Ribosome
Figure 12-39b
Molecular Biology of the Cell
(© Garland Science 2008)
KRM Identification of an Integral Membrane Targeting Factor Digest with Elastase
Centrifuge 1 2 3 4 5 6
E-supernate E-KRM pellet
MW (kd) 25 22 8
Lanes:
1. No additions 2. SRP Only 3. SRP + KRM /digest with Protease 4. SRP + E-KRM 5. SRP + E-Supernate 6. SRP + E-KRM + E Supernate
Identification of SRP Receptor KRM Detergen t Solubilize SRP Affinity Column SRP Receptor
MW (kd) 1 2 3 25 22
Lanes:
1. No additions 2. SRP 3. SRP + SRP Receptor 8
Structure of the RER Translocation Channel (Sec 61 Complex)
Single-Pass 10 TMS Single-Pass Figure 12-42
Molecular Biology of the Cell
(© Garland Science 2008)
(Side-View) (From 2-D EM Images) (Lumenal View) Figure 12-43
Molecular Biology of the Cell
(© Garland Science 2008)
A Single Ribosome Binds to a Sec61 Tetramer
Post-Translational Translocation is Common in Yeast and Bacteria
Figure 12-44
Molecular Biology of the Cell
(© Garland Science 2008) SecA ATPase functions like a piston pushing ~20 aa’s into the channel per cycle
Classification of Membrane Protein Topology
Single-Pass, Bitopic Multipass, Polytopic
Generation of a Type I Single-Pass Topology
Figure 12-46
Molecular Biology of the Cell
(© Garland Science 2008)
Post-translational Translocation Figure 12-47
Molecular Biology of the Cell
(© Garland Science 2008) Type II
Generation of Type II and Type III Single Pass Topologies
Type III
Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences
Type IVa + – + – + – + – Figure 12-48
Molecular Biology of the Cell
(© Garland Science 2008)
Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences
– + Type IVb Figure 12-49
Molecular Biology of the Cell
(© Garland Science 2008)
NH 2 +
The Charge Difference Rule for Multispanning Membrane Proteins
COOH – – + – COOH – + NH 2 NH 2 + –
cytoplasm
NH 2 – + – + COOH + + COOH
cytoplasm
NH 2 +
Transmembrane Charge Inversion Disrupts Local Membrane Topology in Multipass Proteins L1 L3 1
–
L1 2 L2
+
3
–
L3 4
+ COOH
1 2 3 4 L2
cytoplasm
NH 2
L2
COOH
2 3
NH 2 +
1
–
L1 2
–
L2 3
–
L3 4
+ COOH
1
cytoplasm
NH 2
L1 L3 4
COOH
N-Linked Oligosaccharides are Added to Nascent Polypeptides in the Lumen of the RER
Figure 12-51
Molecular Biology of the Cell
(© Garland Science 2008)
Biosynthesis of the Dolichol-P Oligosaccharide Donor
Structure of the High-Mannose Core Oligosaccharide
Processing of the High-Mannose Core Oligosaccharide in the RER
Oligosaccharide Processing in the RER is Used for Quality Control
Figure 12-53
Molecular Biology of the Cell
(© Garland Science 2008)
Disulfide Bridges are Formed in the RER by Protein Disulfide Isomerase (PDI)