The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Tuesday 9/1 2015 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 12-36c Molecular Biology of the Cell (© Garland Science 2008)
Targeting information resides in the Nascent polypeptide chain Ribosomal Subunits are Shared Between Free and Membrane-Bound Polysomes Targeting information resides in the Nascent polypeptide chain Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)
Signal-Mediated Targeting to the RER
Properties of Secretory Signal Sequences cleavage 8-12 Residues ++ Mature Protein N Hydrophobic Core 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 Low MW components Energy (ATP, creatine-P, creatine kinase) Reticulocyte or wheat germ lysate
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* SDS PAGE Protein*
In Vitro Translation of Prolactin mRNA Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary SDS Gel Lanes: 1 2 3 4 5 6 7 8 Purified prolactin No RM RM No RM /digest with Protease RM /digest with Protease RM /detergent treat and add Protease Prolactin mRNA minus SS + RM /digest with Protease SS-globin mRNA + RM /digest with Protease MW (kd) 25 22 18
Identification of a Soluble RER Targeting Factor RM + 0.5 M KCl Centrifuge Supernate = KCl wash Pellet = KRM 1 2 3 4 5 MW (kd) Lanes: No additions KRM KRM / digest with Protease KRM + KCl wash KRM + KCl wash / digest with Protease 25 22 18 8
Purification of the Signal Recognition Particle (SRP) Hydrophobic Chromatography KCl Wash SRP 1 2 3 4 5 MW (kd) Lanes: No additions KRM KRM /digest with Protease KRM + KCl wash /digest with Protease KRM + SRP /digest with Protease 25 22 18 8
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)
Identification of an Integral Membrane Targeting Factor KRM Digest with Elastase Centrifuge E-supernate E-KRM pellet 1 2 3 4 5 6 MW (kd) Lanes: No additions SRP Only SRP + KRM /digest with Protease 25 22 SRP + E-KRM SRP + E-Supernate SRP + E-KRM + E-Supernate 8
Identification of SRP Receptor SRP Affinity Column Detergent Solubilize SRP Receptor KRM 1 2 3 MW (kd) Lanes: No additions SRP SRP + SRP Receptor 25 22 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)
A Single Ribosome Binds to a Sec61 Tetramer (Side-View) (From 2-D EM Images) A Single Ribosome Binds to a Sec61 Tetramer (Lumenal View) Figure 12-43 Molecular Biology of the Cell (© Garland Science 2008)
Post-Translational Translocation is Common in Yeast and Bacteria SecA 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)
Generation of Type II and Type III Single Pass Topologies 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 COOH – + – + – NH2 COOH + cytoplasm + NH2 NH2 – – + – – + NH2 COOH + cytoplasm + COOH
Transmembrane Charge Inversion Disrupts Local Membrane Topology in Multipass Proteins + – + + – NH2 1 2 3 4 COOH 1 2 3 4 L1 L2 L3 L2 cytoplasm NH2 COOH L2 2 3 L1 L3 + – – – + NH2 1 2 3 4 COOH 1 4 L1 L2 L3 cytoplasm NH2 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)