1. The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway Tuesday 9/ Mike Mueckler

2. Ribosome Structure
Figure Molecular Biology of the Cell (© Garland Science 2008)

3. Formation of Polyribosomes
Figure Molecular Biology of the Cell (© Garland Science 2008)

4. 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

5. Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)

6. 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)

7. Signal-Mediated Targeting to the RER

8. 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

9. 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

10. Isolation of Rough Microsomes by Density Gradient Centrifugation
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)

11. In Vitro Translation/Translocation System
mRNA +
Translation Components
Amino acid*
SDS
PAGE
Protein*

12. In Vitro Translation of Prolactin mRNA
Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary
SDS Gel
Lanes:
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

13. Identification of a Soluble RER Targeting FactorRM +
0.5 M KCl
Centrifuge
Supernate = KCl wash
Pellet = KRM
MW (kd)
Lanes:
No additions
KRM
KRM / digest with Protease
KRM + KCl wash
KRM + KCl wash / digest with Protease 25 22 18 8

14. Purification of the Signal Recognition Particle (SRP)
Hydrophobic
Chromatography
KCl Wash
SRP
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

15. 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

16. Structure of the Signal Recognition Particle
(7SL RNA)
Figure 12-39a Molecular Biology of the Cell (© Garland Science 2008)

17. Interactions Between SRP and the Signal Sequence and Ribosome
Figure 12-39b Molecular Biology of the Cell (© Garland Science 2008)

18. Identification of an Integral Membrane Targeting Factor
KRM
Digest with Elastase
Centrifuge
E-supernate
E-KRM pellet
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

19. Identification of SRP Receptor
SRP Affinity Column
Detergent Solubilize
SRP Receptor
KRM
MW (kd)
Lanes:
No additions
SRP
SRP + SRP Receptor 25 22 8

20. Structure of the RER Translocation Channel (Sec 61 Complex)
Single-Pass
10 TMS
Single-Pass
Figure Molecular Biology of the Cell (© Garland Science 2008)

21. 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 Molecular Biology of the Cell (© Garland Science 2008)

22. 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 Molecular Biology of the Cell (© Garland Science 2008)

23. Classification of Membrane Protein Topology
Single-Pass, Bitopic
Multipass, Polytopic

24. Generation of a Type I Single-Pass Topology
Figure Molecular Biology of the Cell (© Garland Science 2008)

25. Generation of Type II and Type III Single Pass Topologies
Post-translational
Translocation
Figure Molecular Biology of the Cell (© Garland Science 2008)

26. Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences
Type IVa + – + + + – – –
Figure Molecular Biology of the Cell (© Garland Science 2008)

27. Multipass Topologies are Generated by Multiple Internal Signal/Anchor Sequences
Type IVb + –
Figure Molecular Biology of the Cell (© Garland Science 2008)

28. The Charge Difference Rule for Multispanning Membrane Proteins
COOH – + – + –
NH2
COOH +
cytoplasm +
NH2
NH2 – – + – – +
NH2
COOH +
cytoplasm +
COOH

29. 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

30. N-Linked Oligosaccharides are Added to Nascent Polypeptides in the Lumen of the RER
Figure Molecular Biology of the Cell (© Garland Science 2008)

31. Biosynthesis of the Dolichol-P Oligosaccharide Donor

32. Structure of the High-Mannose Core Oligosaccharide

33. Processing of the High-Mannose Core Oligosaccharide in the RER

34. Oligosaccharide Processing in the RER is Used for Quality Control
Figure Molecular Biology of the Cell (© Garland Science 2008)

35. Disulfide Bridges are Formed in the RER by Protein Disulfide Isomerase (PDI)