The Signal Hypothesis and
the Targeting of Nascent
Polypeptides to the
Secretory Pathway
Tuesday 9/2 2014
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)
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
8-12 Residues
++
N
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
•
•
•
•
•
•
•
mRNA
Rough microsomes
Ribosomes
tRNAs
Reticulocyte or
wheat germ lysate
Soluble translation factors
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
Lanes:
1.
2.
3.
4.
5.
25
22
6.
7.
18
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
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
KRM / digest with Protease
KRM + KCl wash
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
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%
ribosomes + polyribosomes
cytoplasm
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
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
Detergen
t
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 Sec61 Tetramer
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)
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
–
NH2 +
–
+
–
COOH
COOH
+
NH2
NH2
+
cytoplasm
–
–
+
–
+ COOH
+
NH2
–
+ cytoplasm
COOH
Transmembrane Charge Inversion Disrupts Local
Membrane Topology in Multipass Proteins
L1
NH2 +
–
1
+
2
–
3
L2
L1
+
4
COOH
L3
1
2
L2
NH2
L1
1
–
L1
2
–
L2
3
–
L3
4
+
COOH
COOH
L2
2
+
4
L3
cytoplasm
NH2
3
1
3
L3
4
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)