Bio 103 Winter 2005 Lectures 14

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Bio 103 Winter 2005Lectures 1-4

• Intro and Membrane Transport

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DNA Microarrays

• DNA microarrays are used to map a global pattern
  of gene expression.
• Can compare mRNA levels of thousands of genes at
  one time
• Control versus specific experimental setup

3
Metric System

• 1M adult human
• 1x10-1 decimeter mice
• 1x10-2 centimeter large insects
• 1x10-3 millimeter small insects
• 1x10-6 micrometer cells
• 1x10-9 nanometer small molecules

4
Lipid Structure
What is the significance of double bonds in the
hydrophobic tails?
5
Lipids Composition Affects Membrane Structure
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Lipid Rafts
Why are lipid rafts important to the cell?
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Membrane Interacting Proteins
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Membrane Transport Systems
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Terms of Transport

• Active Transport requires ATP to move substance
  against gradient
• Passive Diffusion No energy input required, no
  proteins required
• Facilitated Diffusion Same as passive but with
  help of a transport protein

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ATP-Pumps

• P-Class Pump
• ABC Superfamily

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Na/K ATPase Pump (P-class)
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ABC Superfamily

• MDR-1 is a member of the ABC Superfamily of pumps

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Types of Transporters
Why do transporters have a Vmax, unlike passive
diffusion?
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Channels Non-gated K Channel
Why cant sodium pass through the potassium
channel, even though sodium is a smaller atom?
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Non-Gated K Channels Cont.

• These channels are almost always open
• They create the membrane potential
• This is why the resting potential across the
  membrane is closer to the potassium potential
  than the sodium potential.

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Gated Ion Channels
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Voltage Gated Channels
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Water (Aquaporin) Channels
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Summary of MembraneTransport
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Putting it All Together
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Triggering of heart beat
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Lectures 5-6

• Nucleus
• Mitochondria

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Review
Lecture 5 Cellular Compartmentalization,
Nuclear Structure and Protein
Transport Lecture 6 Mitochondrial Function and
Protein Transport
Li Zhang
Bio103
Winter 2005
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Lecture 5 Cellular Compartmentalization, Nuclear
Structure and Protein Transport

• I. Cellular Compartmentalization
• Eukaryotic cell is subdivided into
  functionally distinct,
• membrane-enclosed compartments.
• Increased membrane
  surface area
• Specialization of
  functions
• II. Topological Relationships determined by
  Evolution
• (Lecture 5, Slides 6, 7 and 8)
• Nucleus (invagination of the plasma membrane) ?
  Cytosol
• ER, Golgi, Endosomes, Lysosomes, other secretory
  vesicles ? the exterior of the cell
• Mitochondria originated from bacteria cell
  engulfed by pre-eukaryotic cell
• Note a) Topologically equivalent to the
  outside of the cell
• b) Involved in Transmembrane
  protein transport (cyto-exo)
• c) But the lumen of
  mitochondria is unique and remains isolated from
  the membrane traffic that interconnects the
  lumens of many other intracellular compartments.
  

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III. Protein sorting
Proteins mostly synthesized in the cytosol
Proteins with no sorting signal? Remain in the
cytosol Proteins with sorting signal ?
delivered to different compartments
Proteins move between compartments in three
different ways
1. Gatedcyto to cyto 2. Transmembrane cyto
to exo 3. Vesicular exo to exo
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IV. Structure of Nucleus and Mitochondria
The only two organelles that have double
membranes

• Nuclear Pore Complex (NPC) (lecture 5, slide
  14)
• Huge protein complex made of nucleoprorins
• Octagonal symmetry
• Eight filaments extend into cytoplasm
• Nuclear basket extend into nucleoplasm
• A large aqueous pore where the outer and inner
  membrane
• of the nucleus converge
• Greater numbers of NPC? More Active the nucleus
  in transcription

Cytosol
nucleoplasm
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V. Nuclear Transport

• Free Diffusion --- Ions, Small metabolite and
  Globular pr. lt 60kD
• a) Each pore complex contains one or more
  open aqueous channels
• through which small, water-soluble molecules
  can pass
• b) Small molecules small molecules may also
  diffuse between columnar subunits
• Active Transport --- large proteins and
  macromolecular complexes
• Occurs through NPC? Different from the mechanism
  of protein transport
• across
  membranes of other organelles
• Nuclear proteins can transport through a pore
  complex in fully folded conformation
• Gated transport----cyto to cyto

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Nuclear Import

• Why?
• a) Proteins required for nuclear functions
  synthesized in the cytoplasm
• b) During cell division, nuclear envelop
  breaks down and nuclear components need
• to be redelivered after reformation of
  nuclear envelop.
• 2. Discovery of Nuclear-Localization Signal
  (NLS) (lecture 5, slide 19)
• Normal SV40 ? T-antigen localized in the
  nucleus in virus-infected cells
• Mutants of SV40 ? Large T-antigen
  accumulated in the cytoplasm
• Amino acid sequence analysis discovered a
  mutation in the seven-residue sequence
• at the C-terminal of the abnormal protein.
• Fusion of the T-Antigen NLS sequence to a
  cytosolic protein ( pyruvate kinase)
• results in nuclear import of cytosolic
  protein
• NLS can be located anywhere in the protein
  sequence.
• Most NLS rich in basic amino acids such as
  lysine and arginine---there are exceptions
• NLS is not cleaved after protein is imported
  into the nucleus

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Mechanism of Import (lecture 5, slide 23)
Lodish, Molecular Cell Biology, fifth edition,
Fig 12-21, page 511
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Nuclear Export

• Nuclear-Export Signal (NES)
• Exportins recognize and bind to NES.
• Proteins that have both NLS and NES shuffles
  between nucleus and cytoplasm

Lodish, Molecular Cell Biology, fifth edition,
fig 12-23, page 513
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Similarities and Differences between Import and
Export Processes
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Why is the import and export Unidirectional? The
gradient of the two conformational forms of Ran
drives nuclear transport in the appropriate
direction.
GAP
Cytosol
Ran-GDP
GEF
Nucleus
Ran-GTP
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Lecture 6 Mitochondrial Function and Protein
Transport
I. Mitochondrial Function 1. Mitochondria is the
principle site of ATP production 2. How is ATP
produced Under aerobic conditions? 1)
Macromolecules ? monomeric subunits These
enzyme digestion occurs in the intestine outside
cells or in lysosomes 2) Monomeric subunit?
acetyl-CoA Production of limited amounts of
ATP and NADH 3) Citric acid cycle 1 acetyl CoA
? CO2 H2O 3 NADH 1 FADH2 1 GTP This
occurs in the mitochondria matrix 4) Electron
Transport Chain and Proton Pumping
Mitochondria inner membrane. Creates a proton
gradient---PMF 5) ATP synthase and ATP production
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Lecture 6 slide 12, 14, 19

• Two Roles of PMF
• ATP synthesis
• Exchange of ATP with ADP and Pi

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ATP Synthase and ATP Production

• How to prove ATP synthesis requires pH gradient
  across
• the inner membrane? Fig 8-23, page 325
• 2. Structure of ATP Synthase Fig 8-24

Two Multiprotein Complexes F0 F1
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Mitochondrial protein transport
transmembrane (cyto-exo)
1. Energy needed a) ATP needed for removal of
chaperone proteins in the cytosol b)
Translocation requires H electrochemical
gradient, or PMF (proton-motive force) c) ATP
needed for removal of chaperone proteins in the
matrix 2. Mitochondrial targeting sequence
Amphipathic helix 3. Receptor in the outer
membrane 4. Translocons Tom translocon of
the outer membrane Tim translocon of the inner
membrane
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• Mechanism of mitochondrial protein transport
• Protein transport into Matrix
• Protein transport into inner membrane
• Protein transport into the intermembrane space

Fig 16-26, 16-29 and 16-30
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Note a) Matrix targeting sequence is
cleaved after transport b)
Cytosolic chaperones keep proteins in unfolded
state before transport Matrix
chaperones keep proteins unfolded when necessary
c) Matrix targeting sequence is
amphipathic and is cleaved after transport
All other targeting sequences mentioned
are hydrophobic and will be found
in the inner membrane after transport
d) Two roles of PMF ATP synthesis and
transport
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Lectures 7-9

• Protein Sorting

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The Cell
Lectures 7-9 Protein Sorting
ER
Golgi
Lysosomes
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Endoplasmic Reticulum
Rough endoplasmic reticulum (RER) ribosomes
bound to the cytosolic side Smooth endoplasmic
reticulum (SER) no such bound ribosomes
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Protein Sorting ER
http//faculty.washington.edu/kepeter
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Protein Sorting ER

• sequence recognition protein binds to signal
  sequence

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Protein Sorting ER
SRP-ribosome complex binds to the SRP receptor -
GTP (SRP / receptor )
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Protein Sorting ER

• Ribosome is transferred to translocon
• Polypeptide is inserted in the ER

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Protein Sorting ER
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Proteins in ER membrane
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Protein Sorting ER
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Golgi Apparatus
COPII vesicles
Clathrin vesicles (Lysosomes)
COPI vesicles
ER
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GTPases required for Coat Protein Binding

• COPI requires Sar1 GTPase in the membrane
• COPII and Clathrin require ARF

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Vesicles budiing and fusion

• GTP binding proteins (Sar1, ARF) initiated
• Coat proteins bind membrane proteins carrying
  cargo
• Vesicle pinches off
• Coat protein is lost when GTP is hydrolyzed
• v-SANRE exposed and binds t-SNARE
• Fusion

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Retrograde transport Golgi to ER

• Retrieve missorted proteins and v-SNARE
• KDEL sequence on protein recognized by KDEL
  receptor
• Receptor affinity is high in Golgi and low in ER

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Clathrin

• Triskelion
• 3 heavy and 3 light chains
• -36 triskelions form sphere
• -AP protein required

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Transport of Hydrolases Golgi to Lysosome

• Mannose-6-P added to N-linked sugar
• M6P receptor recognizes M6P
• Clathrin/AP mediated
• Enzyme dissociates in acidic pH of endosome

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Lysosomes
pH ?
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Tay-Sachs disease

• Enzyme defective in breakdown
• Gangliosides (glycolipids) accumulate
• Serious consequences
• Blindness
• Death

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Endocytosis

• Endocytosis Endo (within) cytosis (cell) is a
  process in which a substance gains entry into a
  cell without passing through the cell membrane.
  This process is subdivided into three different
  types
• pinocytosis
• phagocytosis / autophagy
• receptor mediated endocytosis

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Receptor Mediated endocytosis LDL (low density
lipoproteins)

• Found in blood
• contains cholesterol
• sphere
• outer boundry is phospholipid
• core of the sphere holds cholesterol
• Inserted into the phospholipid are proteins
  called apoB proteins
• LDL receptors on cell surface bind apoprotein

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Lectures 10-14

• Cytoskeleton
• Tissue formation/cellular interactions

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Microfilaments Actin
G-Actin F-Actin
Cell shape force for cell movement
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Actin Polymerization Critical Concentration
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(No Transcript)
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Regulation of Polymerization
Arp based polymerization create force to push
membrane forward
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Regulation of Polymerization
CapZ
Cell shape
thymosin inhibits, profilin promotes assembly
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Myosin-Powered Cell Movement
Walking toward the end
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Intermediate Filaments
7nm 10nm 24nm
Lamins, keratins, type III, neurofilaments
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Microtubule Subunit
-end
end
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Tubulin Polymerization Dynamic Instability
Fast rate of polymerization
Slow rate of pol, high rate of hydrolysis
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Kinesin Dyenin Motor Proteins
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Cell-Cell Cell-Matrix Interactions
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ECM of Non-Epithelial Tissues Collagen Elastin
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ECM of Epithelial TissuesIntegrins Fibronectins