Signal Hypothesis - Bio 5068

download report

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)