MCB Test 1 Review
Alex Miranda
[email protected]
The Structure of
Biological Membranes
Thursday 9/1/2016
Mike Mueckler
[email protected]
Functions of Cellular Membranes
1. Plasma membrane acts as a selectively permeable barrier to the
environment
• Uptake of nutrients
• Waste disposal
• Maintains intracellular ionic milieu
2. Plasma membrane facilitates communication
• With the environment
• With other cells
3. Intracellular membranes allow compartmentalization and
separation of different chemical reaction pathways
• Increased efficiency through proximity
• Prevent futile cycling through separation
• Protein secretion
Fatty Acid Saturation
Bilayers are Thermodynamically Stable Structures Formed from
Amphapathic Lipids
(Hydrophobic)
(Hydrophilic)
Figure 10-11a Molecular Biology of the Cell (© Garland Science 2008)
Hydrophobic effect in bilayer formation
PhosphoLipid Movements within Bilayers
(µM/sec)
(1012-1013/sec) (108-109/sec)
Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)
A “Scramblase” Enzyme
Catalyzes Symmetric
Growth of Both Leaflets
in the ER
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
A “Flippase” Enzyme
promotes Lipid
Asymmetry in the
Plasma Membrane
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
Phospholipids are Involved in Signal Transduction
1. Activation of Lipid Kinases
PI-4,5P
Figure 10-17a Molecular Biology of the Cell (© Garland Science 2008)
PI-3,4,5P
2. Phospholipases Produce Signaling
Molecules via the Degradation of
Phospholipids
3 Ways in which Lipids May be Transferred Between Different
Intracellular Compartments
Vesicle Fusion
Direct Protein-Mediated Soluble Lipid
Transfer
Binding Proteins
The 3 Basic Categories of Membrane Protein
GPI Anchor
Single-Pass
Multi-pass
b-Strands
Transmembrane
Helix
Linker
Domain
Fatty acyl
anchor
Integral
Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)
LipidAnchored
Peripheral
(can also interact
via PL headgroups)
Membrane Domains are “Inside-Out”
Right-Side Out Soluble Protein
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
Functional Characterization of
Integral Membrane Proteins
Requires Solubilization and
Subsequent Reconstitution
into a Lipid Bilayer
Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008)
The Signal Hypothesis and
the Targeting of Nascent
Polypeptides to the
Secretory Pathway
Tuesday 9/6/2016
Mike Mueckler
[email protected]
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
Multipass Topologies are Generated by
Multiple Internal Signal/Anchor Sequences
Type IVa
+
–
+
+
+
–
–
–
Figure 12-48 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
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)
Oligosaccharide Processing in the RER is Used
for Quality Control
Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)
Thursday 9/4 2014
Mike Mueckler
[email protected]
Proteins are Incorporated Into Mitochondria
Via Several Different Routes
Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008)
Protein Import into the Matrix Requires ATP Hydrolysis and an
Intact Proton Gradient
Across the Inner Membrane
Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008)
Targeting to the Inner Membrane Occurs Via 3 Distinct Routes
Stop-Transfer-Mediated
Single-Pass
Proteins
Oxa1-Mediated
Tom70/Tim22/54-Mediated
Multi-Pass Proteins
ADP/ATP Antiporter
Cytochrome oxidase
subunit CoxVa
ATP Synthase Subunit 9
Targeting to the Outer Membrane
Via the SAM Protein Complex
(Sorting and
Assembly
Machinery)
(b-Barrell)
Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear
Transport
•Bidirectional
•Single Large Pore Complex
Spans 2 lipid bilayers
•Nuclear Pores much larger
than other translocons
Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear Import and Export Sequences are
Recognized by Different Members of the
Same Receptor Family (Keryopherins)
Figure 12-13 Molecular Biology of the Cell (© Garland Science 2008)
Directionality is Conferred on Nuclear Transport by a
Gradient of Ran-GDP/GTP Across the Nuclear Envelope
Figure 12-14 Molecular Biology of the Cell (© Garland Science 2008)