THIS PART OF THE REVIEW COVERS LECTURES BY DRS MUECKLER,
MAHJOUB AND COOPER (TOTAL OF 5 LECTURES) EVERYTHING TESTED ON THE
IN-CLASS PORTION OF THE EXAM FOR THE ABOVE LECTURES IS COVERED IN
THIS REVIEW. HOWEVER, NOT EVERYTHING COVERED IN THIS REVIEW WILL BE
ON THE EXAM. MATERIAL FROM THIS SECTION OF THE REVIEW WILL COUNT
FOR 55 OF THE 100 TOTAL POINTS ON THE EXAM MCB EXAM 1 REVIEW :
SEPTEMBER 27 2014
Slide 2
of The Structure of Biological Membranes
Slide 3
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
Slide 4
Figure 10-2 Molecular Biology of the Cell ( Garland Science
2008) Structure of Phosphoglycerides All Membrane Lipids are
Amphipathic
Slide 5
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 G = S
Slide 6
Slide 7
Figure 10-8 Molecular Biology of the Cell ( Garland Science
2008) The Formation of Cell-Like Spherical Water-Filled Bilayers is
Energetically Favorable
Slide 8
Figure 10-11b Molecular Biology of the Cell ( Garland Science
2008) PhosphoLipid Movements within Bilayers (M/sec) (10 12 -10 13
/sec) (10 8 -10 9 /sec)
Slide 9
Figure 12-57 Molecular Biology of the Cell ( Garland Science
2008) Phosphoglyceride Biosynthesis Occurs at the Cytoplasmic Face
of the ER
Slide 10
Figure 12-58 Molecular Biology of the Cell ( Garland Science
2008) A Scramblase Enzyme Catalyzes Symmetric Growth of Both
Leaflets in the ER
Slide 11
Figure 10-16 Molecular Biology of the Cell ( Garland Science
2008) The Two Plasma Membrane Leaflets Possess Different Lipid
Compositions Enriched in PC, SM, Glycolipids Enriched in PE, PS,
PI
Slide 12
Figure 12-58 Molecular Biology of the Cell ( Garland Science
2008) A Flippase Enzyme promotes Lipid Asymmetry in the Plasma
Membrane Flippases are P-type ATPases
Slide 13
Figure 10-5 Molecular Biology of the Cell ( Garland Science
2008) C12 How Cholesterol Integrates into a Phospholipid Bilayer 1)
INCREASE MEMBRANE FLUIDITY 2) PART OF LIPID RAFTS (SIGNALING
FUNCTION)
Slide 14
3 Ways in which Lipids May be Transferred Between Different
Intracellular Compartments Vesicle Fusion Direct Protein-Mediated
Transfer Soluble Lipid Binding Proteins
Slide 15
Figure 3-5 Molecular Biology of the Cell ( Garland Science
2008) Membrane Domains are Inside-Out Right-Side Out Soluble
Protein
Slide 16
Figure 10-29b Molecular Biology of the Cell ( Garland Science
2008) Detergents Exist in Two Different States in Solution
(Critical Micelle Concentration) CMC DETERMINANTS 1)CHARGE (IONINC
HAS HIGHER CMC THAN NONIONIC) 2)TEMPERATURE 3)CONCENTRATION
Slide 17
Detergent Solubilization of Membrane Proteins Desirable for
Purification of Integral Membrane Proteins
Slide 18
Figure 10-22b Molecular Biology of the Cell ( Garland Science
2008) Transmembrane Domains Can Often be Accurately Identified by
Hydrophobicity Analysis CRITERIA: 1)POSITIVE PEAK 2)GREATER THAN 20
AMINO ACIDS IN LENGTH
Slide 19
The Signal Hypothesis and the Targeting of Nascent Polypeptides
to the Secretory Pathway DR MUECKLER LECTURE 2
Slide 20
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
Slide 21
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
Slide 22
Signal-Mediated Targeting to the RER
Slide 23
Properties of Secretory Signal Sequences Hydrophobic Core N
Mature Protein 8-12 Residues 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 cleavage
Slide 24
In Vitro Translation of Prolactin mRNA Prolactin is a
polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary 1 2
3 4 5 6 7 8 MW (kd) 25 22 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
18 SDS Gel
Slide 25
Figure 12-39b Molecular Biology of the Cell ( Garland Science
2008) Interactions Between SRP and the Signal Sequence and
Ribosome
Slide 26
Figure 12-44 Molecular Biology of the Cell ( Garland Science
2008) Post-Translational Translocation is Common in Yeast and
Bacteria SecA ATPase functions like a piston pushing ~20 aas into
the channel per cycle
Figure 12-51 Molecular Biology of the Cell ( Garland Science
2008) N-Linked Oligosaccharides are Added to Nascent Polypeptides
in the Lumen of the RER
Slide 29
Disulfide Bridges are Formed in the RER by Protein Disulfide
Isomerase (PDI)
Slide 30
MUECKLER LECTURE 3
Slide 31
Mitochondrial Biogenesis Mitochondria contain their own genome
and protein synthetic machinery (tRNAs, mRNAs, ribosomes,
initiation and elongation factors, etc.) Mitochondria are comprised
of hundreds of distinct proteins, only a handful of which are
encoded in the mitochondrial genome (varies by species) Most
mitochondrial proteins are encoded in nuclear DNA, synthesized in
the cytosol, and imported post-translationally into the
organelle
Slide 32
Protein Import into the Matrix Requires Passage Through Two
Separate Membrane Translocons
Slide 33
Proteins Traverse the TOM and TIM Translocons in an Unfolded
State
Slide 34
Figure 12-26 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
Slide 35
Targeting to the Inner Membrane Occurs Via 3 Distinct Routes
Oxa1-MediatedStop-Transfer-MediatedTom70/Tim22/54-Mediated
Multi-Pass Proteins Single-Pass Proteins Cytochrome oxidase subunit
CoxVa ATP Synthase Subunit 9 ADP/ATP Antiporter
Slide 36
Cytochrome B2Cytochrome c Heme Lyase Targeting to the
Intermembranous Space Occurs Via Two Distinct Pathways Direct
Delivery IM Space Protease
Slide 37
Figure 12-27 Molecular Biology of the Cell ( Garland Science
2008) Targeting to the Outer Membrane Via the SAM Protein Complex (
S orting and A ssembly Machinery) ( -Barrell)
Slide 38
Figure 12-14 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
Slide 39
Figure 12-15 Molecular Biology of the Cell ( Garland Science
2008) Nuclear Import and Export Operate Via Reciprocal Use of the
Ran-GDP/GTP Concentration Gradient
Slide 40
Microtubules and their functions in cells. DR. MAHJOUBS
LECTURE
Slide 41
Microtubule Structure Cross-section Hollow tube 24 nm wide ~13
protofilaments Helical structure Polar Plus ends generally distal
Minus ends generally proximal (at MTOC) Composed of Tubulin
Heterodimer
Slide 42
Microtubule Structure & Assembly
Slide 43
The Master Nucleator -TuRC and microtubule nucleation
Slide 44
Microtubule Motors Definition Microtubule-stimulated ATPase
Motility along MTs Dynein Moves towards Minus End of MTs -
Retrograde Kinesin Moves to Plus End of MTs - Anterograde
Slide 45
Microtubules are everywhere Q: How are heterogeneity and
specificity of microtubule function regulated? Different tubulin
isoforms A large number of tubulin PTMs Different motors and
adaptors Diverse MAPs
Slide 46
Organelle Trafficking - ER and Golgi Positioning ER &
Golgi: Golgi near MTOC Minus Ends are at MTOC Golgi Position
Requires Dynein ER Tubular network spread about the cell Kinesin
moves the tubules peripherally
Slide 47
The centrosome as MTOC 1 centrosome per cell 2 centrioles per
centrosome (in interphase) Centriole size is highly conserved
throughout evolution: ~250nm diameter, ~500nm length
Slide 48
Mechanism of centrosome duplication Q: How can a cell make an
exact copy of such an elaborate structure? G1/S SSG2/MS/G2 M/G1
-Templated duplication during the S phase of the cell cycle
Slide 49
Centriole structure establishing 9-fold symmetry Q: How is the
9-fold rotational symmetry of microtubules established during
centriole duplication? Sas6 has intrinsic symmetry when forming
oligomers
Slide 50
Centriole structure establishing 9-fold symmetry Q: But how
come Sas6 doesnt just form a bunch of rings non-specifically in the
cytoplasm? A: - Expression restricted to G1/S phase (temporal
regulation) - Recruited to parental centrioles (spatial
regulation)
Slide 51
Mitotic Spindle Assembly Centrosome duplicates and is
segragated to each side of the nucleus Nuclear envelope breakdown
in prophase MTs rearrange via dynamic instability
Slide 52
Spindle microtubules
Slide 53
Models for Chromosomes Moving to the Pole Treadmilling?
Depolymerization at pole (MTOC) Depolymerization at Kinetochore?
How to remain bound while end shrinks? Motors at Kinetochore or
Pole?
Slide 54
Structure of motile cilium: Cross-section
Slide 55
Conversion of sliding to bending Q: If all dyneins pull
together, how does the axoneme bend back and forth? A: THE ROTATION
OF THE CENTRAL PAIR TURNS ON THE DYNEIN ARMS SEQUENTIALLY TO ALLOW
BENDING TO OCCUR
Slide 56
The primary cilium: non-motile sensory organelle Example: -THE
CILIA STICK INTO THE LUMEN OF THE NEPHRON TO SENSE URINE FLOW
MECHANO-SENSATION
Slide 57
Fundamental processes in cell biology mediated by primary
cilium Extracellular signal Cell division Cell differentiation Cell
migration Cell polarity and organization X X X X X
Slide 58
Biophysics of Helical Polymers Dr. Cooper
Slide 59
Self-Assembly by Proteins - Entropy & the Hydrophobic
Effect High Order in Assembled State Implies Lower Entropy, which
is Unfavorable G = H - TS must be 0, S>>0 ! Higher Entropy
=> Disorder in Assembled State Ordered Water on Hydrophobic
Surface of Protein Subunit is Released
Slide 60
Why Use Subunits to Make Large Molecules? Efficient Use of the
Genome Error Management Variable Size Disassembly / Reassembly
Slide 61
Assembly of Helical Filaments Add & Lose Subunits Only at
Ends ON Rate = k + c 1 N OFF Rate = k - N c 1 = Concentration of
Monomers N = Concentration of Filament Ends
Critical Concentration and Binding Affinity K a = 1 _c1_c1 K d
= [N j+1 ] [N j ] = _c1_c1 _c1_c1
Slide 64
Treadmilling Polar Filaments have Two Different Ends Can Have
Different Critical Concentrations at the Two Ends Steady State
Critical Concentration is an Intermediate Value Net Addition at One
End, Net Loss at the Other End
Slide 65
How do Cells Regulate the Number and Length of Filaments? Limit
Growth Intrinsic to Protein Deplete Subunits Capture by Capping End
Template Create New Filaments Nucleation - End or Side Bolus of
Subunits - High Concentration
Slide 66
Nucleotide Hydrolysis Provides Energy for Dynamic Instability
The Basic Facts... Tubulin Binds GTP or GDP GTP Tubulin Polymerizes
Strongly GDP Tubulin Polymerizes Poorly Subunits Exchange w/ Free
GTP GTP on Tubulin Hydrolyzes to GDP over Time after Addition to
Microtubule
Slide 67
Nucleotide Hydrolysis Provides Energy for Dynamic Instability
At Steady State, at any given time... Most Ends have a GTP Cap and
Grow Slowly A Few Ends Lose their GTP Cap Exposing GDP-tubulin
subunits so the Microtubule Shrinks Rapidly Occurs In Vitro and In
Vivo for Tubulin - Extensive and Relevant
Slide 68
Intermediate Filaments Dr. Cooper
Slide 69
Intermediate Filament Biochemical Properties In Vitro Very
stable. Little subunit exchange. Very strong. Filaments do not
break. MTs strong but brittle Actin weak
Slide 70
Intermediate Filament Structure & Assembly Key Points:
-Monomer and parallel heterodimer have polarity -Antiparallel
tetramer stage is where polarity is lost -No directionality for
motor proteins to use
Slide 71
Regulation of IF Assembly Notoriously Stable No Nucleotide
Filaments Move Little Precursors Move More Disassemble Somewhat
during Mitosis Phosphorylation by Cyclin-dependent Kinase
Slide 72
Vimentin All Cells in Early Development Cage Around Nucleus
Interacts with Mts Vimentin Knockout Mouse Initially normal at
gross inspection Cultured cells have altered properties of
uncertain significance
Slide 73
Desmin Expressed in Muscle Elastic Elements to Prevent
Over-stretching Connects / Aligns Z lines Knockout Mouse - Deranged
Myofibril Architecture
Slide 74
Keratins Expressed in Epithelia Keratin Filaments Connect to
Desmosome and Hemidesmosomes Differentiation of Epidermis includes
Production of Massive Amounts of Keratin Provides Outer Protection
of Skin Composes Hair, Nails, Feathers, etc.
Slide 75
Keratin Mutations are Basis for Human Epidermal Diseases
Structure/Function Analysis of Keratin Assembly Point Mutation in
Terminal Domain Fails to Assemble Mutant is Dominant, even in Low
Amounts, in Cultured Cells and Mice
Slide 76
Neurofilament Neurofilament H, M, L Copolymer Prevent Axon
Breakage Diseases with Clumps of Neurofilaments Superoxide
dismutase model for ALS Clumps are secondary, not causative
Slide 77
Lamins Square Lattice on Inner Surface of Nuclear Membrane
Present in Metazoans (Animals, not Plants or unicellular organisms)
Mitosis Breakdown Phosphorylation of A & C by Cyclin-depen
Kinase B remains with Membrane Mutations Cause Accelerated Aging
Diseases Progerias - Dominant Mutations