MCB: Exam 1 Review
Mueckler
• The Structure of Biological Membranes
• The Signal Hypothesis and the Targeting of
Nascent Polypeptides to the Secretory
Pathway
• Targeting of Nascent Polypeptides Outside of
the Secretory Pathway
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
Mueckler1_MembraneStructure
Composition of Animal Cell Membranes
• Hydrated, proteinaceous lipid bilayers
• By weight: 20% water, 80% solids
• Solids:
Lipid
~90%
Protein
Carbohydrate (~10%)
• Phospholipids responsible for basic membrane
bilayer structure and physical properties
• Membranes are 2-dimensional fluids into which
proteins are dissolved or embedded
Mueckler1_MembraneStructure
Structure of Phosphoglycerides
All Membrane Lipids are Amphipathic
Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
Bilayers are Thermodynamically Stable
Structures Formed from Amphathic Lipids
(Hydrophobic)
(Hydrophilic)
Figure 10-11a Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
Lipid Bilayer Formation is Driven by
the Hydrophobic Effect
• HE causes hydrophobic surfaces such as fatty
acyl chains to aggregate in water
• Water molecules squeeze hydrophobic
molecules into as compact a surface area as
possible in order to the minimize the free
energy state (G) of the system by maximizing
the entropy (S) or degree of disorder of the
water molecules
• DG = DH  TDS
Mueckler1_MembraneStructure
Mueckler1_MembraneStructure
Lipid Bilayer Formation is a
Spontaneous Process
Gently mix PL and water
Vigorous mixing
Figure 1-12 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008)
Mueckler1_MembraneStructure
The Formation of Cell-Like
Spherical Water-Filled
Bilayers is Energetically
Favorable
Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
Phosphoglycerides are Classified by their Head Groups
Phosphatidylethanolamine
Phosphatidylcholine
Phosphatidylserine
Ether Bond at C1
Phosphatidylinositol
PS and PI bear a net negative charge
at neutral pH
Mueckler1_MembraneStructure
PhosphoLipid Movements within Bilayers
(µM/sec)
(1012-1013/sec) (108-109/sec)
Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
Pure Phospholipid Bilayers Undergo Phase Transition
Gauche Isomer Formation
All-Trans
Below Lipid Phase Transition Temp
Above Lipid Phase Transition Temp
Mueckler1_MembraneStructure
A “Scramblase” Enzyme
Catalyzes Symmetric
Growth of Both Leaflets
in the ER
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
The Two Plasma Membrane Leaflets
Possess Different Lipid Compositions
Enriched in PC, SM, Glycolipids
Enriched in PE, PS, PI
Figure 10-16 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
A “Flippase” Enzyme
promotes Lipid
Asymmetry in the
Plasma Membrane
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
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
Mueckler1_MembraneStructure
2. Phospholipases Produce Signaling
Molecules via the Degradation of
Phospholipids
Mueckler1_MembraneStructure
Phospholipase C Activation Produces Two
Intracellular Signaling Molecules
Diacylglycerol
PI-4,5P
I-1,4,5P
Figure 10-17b Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
3 Ways in which Lipids May be Transferred
Between Different Intracellular Compartments
Vesicle Fusion
Direct Protein-Mediated Soluble Lipid
Transfer
Binding Proteins
Mueckler1_MembraneStructure
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)
Mueckler1_MembraneStructure
Membrane Domains are “Inside-Out”
Right-Side Out Soluble Protein
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
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)
Mueckler1_MembraneStructure
Transmembrane a-Helices
H-Bond
1. Right-handed
2. Stability in bilayer results from
maximum hydrogen bonding of peptide
backbone
3. Usually > 20 residues in length (rise of
1.5 Å/residue)
4. Exhibit various degrees of tilt with
respect to the membrane and can bend
due to helix breaking residues
5. Mostly hydrophobic side-chains in
single-pass proteins
6. Multi-pass proteins can possess
hydrophobic, polar, or amphipathic
transmembrane helices
Mueckler1_MembraneStructure
Transmembrane Domains Can Often be Accurately
Identified by Hydrophobicity Analysis
Figure 10-22b Molecular Biology of the Cell (© Garland Science 2008)
Mueckler1_MembraneStructure
Mueckler
• The Structure of Biological Membranes
• The Signal Hypothesis and the Targeting of
Nascent Polypeptides to the Secretory
Pathway
• Targeting of Nascent Polypeptides Outside of
the Secretory Pathway
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
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)
Disulfide Bridges are Formed in the RER
by Protein Disulfide Isomerase (PDI)
Mueckler
• The Structure of Biological Membranes
• The Signal Hypothesis and the Targeting of
Nascent Polypeptides to the Secretory
Pathway
• Targeting of Nascent Polypeptides Outside of
the Secretory Pathway
Proteins are Incorporated Into Mitochondria
Via Several Different Routes
Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008)
Targeting to the Matrix Requires an NTerminal Import Sequence
N-terminal Import Sequences Form Amphipathic a
Helices that Interact with the Tom20/22 Receptor
Hydrophobic cleft
Figure 12-22 Molecular Biology of the Cell (© Garland Science 2008)
Protein Import into the Matrix
Requires Passage Through
Two Separate Membrane Translocons
Proteins Traverse the TOM and TIM
Translocons in an Unfolded State
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 Intermembranous Space
Occurs Via Two Distinct Pathways
Indirect IM Space Protease
Cytochrome B2
Direct Delivery
Cytochrome c Heme Lyase
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 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)
Nuclear Import and Export Operate Via Reciprocal
Use of the Ran-GDP/GTP Concentration Gradient
Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008)