The Signal Hypothesis and the Targeting of Nascent
- Slides: 35
The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Tuesday 8/30/2018 Mike Mueckler mmueckler@wustl. edu
Ribosome Structure Figure 6 -63 Molecular Biology of the Cell (© Garland Science 2008)
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 -36 c 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 Figure 12 -41 a Molecular Biology of the Cell (© Garland Science 2008)
Signal-Mediated Targeting to the RER
Properties of Secretory Signal Sequences ++ N 8 -12 Residues Hydrophobic Core cleavage 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 • • m. RNA Rough microsomes Ribosomes t. RNAs Reticulocyte or Soluble translation factors wheat germ lysate Low MW components Energy (ATP, creatine-P, creatine kinase)
Isolation of Rough Microsomes by Density Gradient Centrifugation Figure 12 -37 b Molecular Biology of the Cell (© Garland Science 2008)
In Vitro Translation/Translocation System m. RNA + Translation Components + Amino acid* Protein* SDS PAGE
In Vitro Translation of Prolactin m. RNA Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary MW (kd) 25 22 SDS Gel 1 2 3 4 5 6 7 8 Lanes: 1. 2. 3. 4. 5. 6. 7. 18 8. Purified prolactin No RM RM No RM /digest with Protease RM /detergent treat and add Protease Prolactin m. RNA minus SS + RM /digest with Protease SS-globin m. RNA + RM /digest with Protease
Identification of a Soluble RER Targeting Factor RM Centrifuge + 0. 5 M KCl MW (kd) 25 22 18 8 Supernate = KCl wash Pellet = KRM 1 2 3 4 5 Lanes: 1. 2. 3. 4. 5. No additions KRM / digest with Protease KRM + KCl wash / digest with Protease
Purification of the Signal Recognition Particle (SRP) KCl Wash MW (kd) 25 22 18 8 Hydrophobic Chromatography 1 2 3 4 5 SRP Lanes: 1. 2. 3. 4. No additions KRM /digest with Protease 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% 15% 38% Conclusions: ribosomes + polyribosomes cytoplasm rough endoplasmic reticulum • 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 (7 SL RNA) Figure 12 -39 a Molecular Biology of the Cell (© Garland Science 2008)
Interactions Between SRP and the Signal Sequence and Ribosome Figure 12 -39 b Molecular Biology of the Cell (© Garland Science 2008)
Identification of an Integral Membrane Targeting Factor Digest with Elastase KRM MW (kd) 25 22 8 Centrifuge 1 2 3 4 5 6 E-supernate E-KRM pellet 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 + ESupernate
Identification of SRP Receptor Detergent Solubilize KRM MW (kd) 25 22 8 SRP Affinity Column SRP Receptor 1 2 3 Lanes: 1. No additions 2. SRP 3. SRP + SRP Receptor
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 Sec 61 Tetramer
Post-Translational Translocation is Common in Yeast and Bacteria Sec. A ATPase functions like a piston pushing ~20 aa’s into the channel per cycle Figure 12 -44 Molecular Biology of the Cell (© Garland Science 2008)
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)
Type II Generation of Type II and Type III Single Pass Topologies Type III Post-translational Translocation Figure 12 -47 Molecular Biology of the Cell (© Garland Science 2008)
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)
The Charge Difference Rule for Multispanning Membrane Proteins – NH 2 + – COOH + NH 2 – + cytoplasm – + COOH + NH 2 – COOH + cytoplasm COOH
Transmembrane Charge Inversion Disrupts Local Membrane Topology in Multipass Proteins L 1 NH 2 + – 1 + 2 – 3 L 2 L 1 + 4 L 3 COOH L 3 1 2 NH 2 L 1 1 – L 1 2 – L 2 3 – L 3 4 + COOH L 2 2 + 4 L 2 cytoplasm NH 2 3 1 3 L 3 4 cytoplasm NH 2 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)
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