Subcellular Organization and Organelles

1
Subcellular Organization and Organelles

• Goals
• Explain function of various subcellular
  organelles and other structures
• Explain how subcellular components are involved
  in cell signalling

2
Subcellular Organization

• Protein Synthesis
• RER
• Golgi
• Mitochondria
• Cytoskeleton
• Molecular Motors
• Axonal Transport

3
Protein Synthesis

• Differences with other cells due to functionally
  distinct compartments and long distance transport
• Outline
• Types of Proteins
• Protein Translation by RER
• Co-translational modification
• Processing by Golgi
• Post-translational modification
• Other protein processing

4
Types of Proteins

• Integral Membrane Proteins
• Segments embedded in lipid bilayer, or
• Segments covalently bound to membrane molecules
• Type I - N terminus extracellular
• Type II - N terminus cytoplasmic
• Both types are synthesized in RER
• E.g. Ionic channels, synaptic channels

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Types of Proteins
Peripheral
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Types of Proteins

• Peripheral Membrane Proteins
• Cytoplasmic Surface
• Do not cross any membrane during biogenesis
• Interact with membrane by association with
• Phospholipids , or
• Cytoplasmic tails of integral proteins, or
• Affinity for other peripheral proteins
• Synthesized by free polysomes
• E.G. PSD-95, AKAP

7
Translation of IntegralMembrane Proteins

• Translation of mRNA
• Folding into correct structure
• Movement into Golgi
• Processing by Golgi
• Packaging into vesicles

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Protein Translation

• Synthesis of nascent peptide by polysome
• Binding of signal recognition particle (SRP a
  ribonucleoprotein) to emergent hydrophobic signal
  sequence

mRNA
Poly- some
Peptide
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Protein Translation
mRNA

• SRP binding causes translation to stop
• SRP docks with receptor in RER

Poly- some
Peptide
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Protein Translation

• SRP dissociates from signal sequence (GTP
  hydrolyzed to GDP)
• Emerging polypeptide chain translocates into ER
  through translocon

translocon
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Protein Translation

• Translocation Amino acids are threaded through
  aqueous pore of translocon as they are formed
• Protein folded into correct conformation with
  help of molecular chaperones within ER

translocon
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Protein Translation

• Variations for membrane proteins
• Stop signal halts translocation stabilizes
  polypeptide in membrane
• Sequential start and stop signals determines
  topology in membrane (e.g. number of
  transmembrane segments)

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Co-translation modification

• Two modifications performed during translation
  (while still in RER)
• N-terminal hydrophobic signal sequence removed by
  signal peptidase
• Oligosaccharides transferred to side chains of
  asparagine residues glycosylation
• Transferred from lipid carrier dolichol phosphate
• Asparagines must be in N X T sequence
• Oligo is linked to the Asp by two
  N-acetylglucosamine

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Golgi Processing
Free Polysomes for translation of peripheral
proteins
RER
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Golgi Processing

• Two major functions
• Sorts and targets proteins (TGN, CGN)
• Post-translational modifications (stacks)
• Three separate golgi compartments
• Cis-Golgi Network (CGN)
• Golgi stacks
• Cis, medial and trans
• Trans-Golgi Network (TGN)

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Golgi Processing

• CGN receives vesicles of proteins from RER
• Vesicle formation
• Vesicles bud from RER
• Several types of proteins assemble around bud
• Coat Proteins (COP) assemble into coatamer
• GTP binding protein called ADP-ribosylation
  factor (ARF)
• AP-1 adaptin recruits coat proteins to membrane
• p200

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Golgi Processing
Transiting Proteins
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Golgi Processing

• Vesicle moves to and docks with Golgi
• Coatamer dissociation triggered by hydrolysis of
  GTP bound to ARF
• GTP hydrolysis caused by GAP (GTPase accelerating
  protein) in Golgi membrane
• ARF re-engergized by Guanine-nucleitide exchange
  factor (GEF) which puts on new GTP

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Golgi Processing

• Fusion to Golgi (similar to transmitter fusion)
• Mediated by
• N-ethylmaleimide-sensitive factor (NSF)
• Soluble NSF attachment proteins (SNAPs)
• SNAP receptors (SNAREs)
• tSNARE - golgi protein
• vSNARE - vesicle protein
• vSNARE associates with tSNARE
• NSF and SNAPS associate with SNARE complex
• Additional details of vesicle fusion later in
  course

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tSnare
COP
vSnare
GTP hydrolysis COP and ARF dissociation
ARF
NSF and SNAP association with Snare complex
Fusion
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Golgi Processing

• Post-translational modification
• Occurs in cis- to trans- stacks
• Modification of existing oligosaccharides
  (attached to asparagines) by glycosidases
• Addition of sugars by glycosyl transferases
• To serine or threonine residues
• Sorting of vesicles in CGN and TGN
• Clathrin coat for late endosome vesicles
• Lacelike coat protein for vesicular transport

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Rab Proteins

• Small GTP binding proteins
• Important for endocytosis and vesicle fusion
• Each stage of endocytosis may have different Rab
  protein
• Rab5a fusion
• Rab6 transport from TGN to endosomes

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Peripheral Membrane Proteins

• Synthesized by free polysomes
• Targeting mechanisms different than integral
  proteins
• mRNA concentrated in discrete cell regions
• Local protein translation
• Free polysomes are associated with cytoskeletal
  structures, not randomly distributed
• Polysomes with mRNAs for MAP2 near proximal
  dendrite
• Polysomes with mRNAs for myelin basic protein
  near oligodendrocyte processes

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Lysosomes

• Membrane bound organelles with high content of
  acid hydrolases
• Function in protein and lipid degradation
• Dysfunction associated with globoid cell
  leukodystrophy, metachromatic leukodystrophy
• Proteins destined for lysosomal function are
  labeled
• Soluble proteins labeled with mannose
  6-phosphate Lysosomes have M6P receptors
• Membrane proteins targeted by cytoplasmic tail
  signals

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Mitochondria

• Have two membranes inner and outer
• Inner membrane is site of oxidative
  phosphorylation
• Electron transfer and ATP synthesis
• Has own circular DNA for some proteins
• Inherited through mother
• Most proteins synthesized in cytoplasm

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Mitochondrial Proteins

• Synthesized on free polysomes
• Partially folded to prevent degradation
• Unfolded proteins are targets of peptidases
• Posttranslation import uses molecular chaperones
  to prevent complete folding
• Hsp70 and hsp60
• Heat shock proteins - upregulated during heat
• Prevent protein conformation changes during heat
  stress

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Mitochondrial Proteins

• Several proteins, e.g. Hsp70, in outer membrane
  form receptor/pore complex
• Complex interacts with inner membrane to minimize
  distance between membranes
• Electron transport produces electrical potential
  that facilitates import
• Hsp70 dissociates from transported protein
• Hsp60 helps with final folding

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Subcellular Organization

• Protein Synthesis
• Cytoskeleton
• Molecular Motors
• Axonal Transport

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Cytoskeleton

• Heterogeneous, dynamic network of filamentous
  structures
• Three components
• Microfilaments (actins)
• Microtubules (tubulins)
• Intermediate filaments
• Not all cells have all types
• Oligodendrocytes have no intermediate filaments

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Cytoskeleton
Myelin
Microtubules
Intermediate filaments
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Cytoskeletal elements
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Function of Microtubules

• Cell movement
• Functional core of cilia and flagella
• Mitotic spindle
• Organelle involved in cell division
• Inhabitants of axons and dendrites
• Intracellular transport
• Essential for fast-axonal transport
• Cell Morphology

33
Microtubules

• Smallest subunit is tubulin
• 10 of total brain protein
• a and b tubulin
• 50 kDa proteins
• Multiple genes for both types
• Different gene products are enriched or specific
  to neurons
• Different gene products are expressed at specific
  times in development

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Microtubules

• Second smallest subunit is "gobule"
• Heterodimer of a and b tubulin
• Protofilaments
• Linear arrangement of globular subunits
• 12-14 protofilaments form microtubule
• 25 nm diameter, hollow tube
• Up to hundreds mm length

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Microtubules
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Microtubule Formation

• Polymerization
• depends on GTP
• is promoted by microtubule-associated proteins
  (MAPs)
• Initially formed (nucleated) at
  microtubule-organizing center
• Contain ?-tubulin which functions as nucleating
  protein
• Subsequently released for delivery to appropriate
  (dendritic vs axonal) compartments
• Involves Katanin, a severing proteins

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Microtubules

• Microtubule orientation
• Polarized (fast-growing) and (slow-growing)
  ends
• Plus () end is distal in axons
• Both polarities seen in dendrites
• Dendrite microtubules are less aligned and less
  regular in spacing
• Half of axonal microtubules are particularly
  stable (mechanism unknown)

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Microtubules

• Post-translation modification (role unknown)
• Phosphorylation (a tubulin)
• Upregulated during neurite outgrowth
• Acetylation-deacetylation (a tubulin)
• Long-lived microtubules tend to be acetylated
• Deactylation upon disassembly
• Tyrosination-detyrosination (b tubulin)
• Synthesized/assembled with Glu-Tyr peptide at C
  terminus
• Detyrosination (with time) leaves Glu-tubulin
• Do not affect stability of microtubules

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Microtubule Associated Proteins (MAPs)

• Multiplicity of types, differentially expressed
• Play a role in stabilizing and orgainzing
  microtubule skeleton
• Identity and phosphorylation state differ between
  dendritic and axonal MAPs
• Axonal microtubules assemble into long,
  continuous structures
• 50 are biochemically distinct and highly stable
• Dendritic microtubules assemble into shorter
  structures

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Microtubule Associated Proteins (MAPs)

• Two heterogenous groups
• Tau proteins
• Polypeptide constituents of neurofibrillary
  tangles
• Bind to microtubules during assembly-disassembly
  cycles
• Promote microtubule assembly and stabilization
• High molecular weight MAPs
• MAP-2 primarily in dendrites
• Side arms protrude from microtubule surface
• Molecular mass gt 1300 kDa

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Summary of Microtubules
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Function of Microfilaments

• Regulation of membrane movement
• Prominent in growth cones (Actin)
• Dynamic changes in dendritic spine morphology
• Muscle Contraction
• In skeletal muscle (actin interacting with
  myosin)
• Local trafficking
• Sensitive to local neuronal environment

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Microfilaments

• Abundant in
• Presynaptic terminals
• Dendritic spines
• Growth cones
• Present throughout cytoplasm
• Actin cytoskelaton is universal in eukaryotes

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Microfilaments

• Two twisted strands of actin subunits
• 4-6 nm diameter
• 20-50 nm length (quite variable)

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Microfilaments

• Multiple actin genes
• a-actin
• Four genes for four muscle types
• b-actin, g-actin
• Abundant in nervous tissue
• All proteins similar (highly conserved)

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Microfilaments - Proteins

• Proteins associated with Microfilaments
• Molecular motors (considered later)
• Monomer actin-binding proteins
• Regulate amount of actin assembled into
  microfilaments by sequestering actin monomers
• Allows rapid mobilization by unbinding
• Capping proteins
• Anchor microfilaments to other structures, e.g.
  membrane proteins
• Regulate microfilament length
• Mutation in Schwann cells causes
  neurofibromatosis 2

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Microfilaments - Proteins

• Cross-linking and bundling proteins
• Create higher-order complexes by bundling
  microfilaments
• Mediate interactions between microfilaments and
  membrane proteins
• E.g. Spectrin plays a role in localization of ion
  channels and receptors
• E.g. Dystrophin clusters receptors
• mutation causes Duchenne muscular dystrophy
• Localize ion and synaptic channels in membrane

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Microfilaments - Proteins

• Gelsolin family
• Function
• Cap barbed end of microfilament
• Sever microfilaments
• Nucleate microfilament assembly
• Importance
• Severing activity is calcium activated
• Alteration of membrane cytoskeleton in response
  to calcium transients
• Activity regulated by 2nd messengers, e.g. PIP2

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Neurofilaments - Function

• Not as clear as microtubules and microfilaments
• Metabolic stability
• Stabilize and maintain neuron morphology
• Disruption of neurofilament organization is
  hallmark of motor neuron disease (e.g. ALS)
• Accumulation of neurofilaments caused by
  alterations in gene expression, or exposure to
  neurotoxins
• Is this causal or effect of disease?

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Neurofilaments
Type IV
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Intermediate filaments

• Previously called neurofilaments
• 8-12 nm diameter
• Many mm length
• Five classes
• Type I, II Keratin (hair and nails)
• Type V nuclear lamins (all nucleated cells)
• Type III, IV neuronal function

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Intermediate Filaments

• Type III
• Include glial fibrillary acidic protein, vimentin
• Molecular weight is 45 60 kDa
• Small amino and carboxy terminal sequences
• Form smooth filaments without side arms
• May be packed tightly
• Restricted to glia and embryonic neurons
• Physiological role is uncertain

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Intermediate Filaments

• Type III
• Peripherin
• Unique to neurons
• Expressed during development and regeneration
• Can coassemble with type IV intermediate filaments

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Intermediate Filaments

• Type IV
• Metabolically very stable
• Expressed only in neurons
• Subunits possess "side arms"
• limits packing density
• Highly phosphorylated in axons
• High density of surface charge limits packing
  density
• Play a role in determining axonal diameter
• Glutamate rich tail region
• Basis for silver stain reaction for neurons

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Intermediate Filaments

• Neurofilament Triplet proteins
• Three types subunits High, medium and low MW
• Only the high (NFH) and medium (NFM) have side
  arms and are phosphorylated
• Primary type of filament formed from all three
  subunits (triple) with varying stoichiometries
• Altered expression associated with degenerative
  disease, similar to ALS
• ?-internexin
• Expressed early in development
• Persists in cerebellar granule cells

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Subcellular Organization

• Protein Synthesis
• Cytoskeleton
• Molecular Motors
• Axonal Transport

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Molecular Motors

• Molecules that hydrolyze ATP (ATPase)
• Drive cell movement such as axonal transport
• Three types
• Myosin
• Muscle contraction via interaction with
  microfilaments
• Dynein
• Kinesin
• Associated with mitosis

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Kinesin

• 40 different genes in 14 subfamilies
• Axonal transport of membrane bound organelles,
  translocation of microtubules
• Strongly inhibited by adenylyl-imidodiphosphate
  (AMP-PNP nonhydrolyzable ATP analog)
• Head contains ATP binding and microtubule binding
  domains
• Kinesin-1 is most abundant in brain and best
  characterized

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Kinesin-1

• Rod shaped, protein, 80 nm in length
• Heterotetramer
• Two heavy chains head and shaft
• 350 conserved amino acids (head region)
  constitute motor domain
• Two light chains fan shaped region
• Divergent part (tail region) of sequence targets
  particular organelle or region of cell
• Synaptic vesicles, mitochondria, coated vesicles,
  lysosomes

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Kinesin

• Some kinesins are
• Monomers (KIF1A)
• Trimers (KIF3A/B)

Head
Tail
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Kinesin

• Responsible for fast axonal transport toward
  distal (terminal) end
• Head attaches to microtubule
• Tail attaches to organelle
• Hydrolysis of ATP moves kinesin head distally,
  toward plus end of microtubule

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Dynein

• Multiple subtypes flagellar and cytoplasmic
• Cytoplasmic dynein is 40 nm in length
• MAP1c is one type
• Retrograde fast axonal transport
• Substrate is long microtubules
• Slow axonal transport
• Microtubules in Anterograde direction
• Substrate is actin filaments or long microtubules
• Weakly inhibited by AMP-PNP

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Dynein Structure
Motor domains
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Myosins

• First identified in skeletal muscles
• 40 different genes comprising 18 subfamilies
• Myosin I, II and V found in nervous system
• Myosin VI and VIIA also in nervous system
• Implicated in congenital deafness
• Function in neuronal growth and development
• Likely role in growth cone motility, synaptic
  plasticity, neurotransmitter release

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Myosin I

• Structure
• Single heavy chain
• Function
• Interacts directly with membrane surfaces
• May generate movement of plasma membrane
  components
• Mechanotransduction (myosin Ic expressed in
  stereocilia of hair cells)

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Myosin II

• Structure
• Dimer composed of two heavy chains
• Two dimers may form bipolar filaments
• Function
• Contractile ring in mitosis
• Unknown role in neurons

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Myosin V

• Structure
• Dimer composed of two heavy chains
• Multiple calmodulin binding sites
• Function
• Found in growth cones
• "Dilute" mutation results in seizures in adult
  mice

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Myosin Structure
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Subcellular Organization

• Protein Synthesis
• Cytoskeleton
• Molecular Motors
• Axonal Transport
• Slow transport
• Fast transport

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Axonal Transport

• Needed because diffusion takes 10 days/cm
• Fast movement alternates with long pauses
• Slow Transport
• Two orders of magnitude slower than fast
• Component a 0.1 to 1 mm/day
• Cytoskeletal proteins, intermediate filaments,
  microtubule proteins (move as assembled polymers)
• Component b 2 to 4 mm/day
• Various polypetides such as actin and enzymes
• Rate limiting for nerve growth or regeneration

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Slow Transport

• Synthesis on free polysomes
• Assemble into microtubules
• Nucleation at microtubule organizing center
• released for migration
• Move via dynein along actin/microfilaments
• Intermediate filaments hitchhike on microtubules
• Delivery

actin
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Slow Axonal Transport

• Motors (dynein) interacts with axonal membrane
  cytoskeleton to move microtubules with plus ()
  end leading
• Minimal degradation of cytoskeleton within axon
• Degradation (at destination only) adjusted to
  produce maintenance or growth
• Slow degradation allows proteins to accumulate

73
Fast Axonal Transport

• Proteins moved as part of cell structure, not as
  individual polypeptides
• Rate is independent of size or molecular weight
• Anterograde Substances (via Kinesin-1)
• Mitochondria
• Neurotransmitters and neuropeptides
• Membrane associated enzymes
• Retrograde Substances (via dynein)
• Similar to Anterograde
• Exogenous materials taken up by terminal,
  endocytosis
• Neurotrophic factors and viral particles (Rabies)

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Fast Axonal Transport

• Polypeptides synthesized on RER
• Processed and packaged in Golgi
• Appropriate motors associate with vesicles,
  mitochondria
• Transported down axon 100-400 mm/day
• Released at appropriate place

75
Fast Retrograde Transport

• Proteins taken up by clathrin coated pits
• Vesicles taken up by endocytic pathway
• Structures are sorted for recycling versus
  transport
• Appropriate dynein attach to vesicles
• Become lysosomes on reaching soma

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Trafficking

• Targeting of different proteins to different
  compartments
• Sodium channels to Nodes of Ranvier
• Neurotransmitters to axon terminals
• Regulated by phosphorylation / dephosphorylation
  reactions
• Many proteins, e.g. enzymes channels, modulated
  by phosphate groups

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Trafficking
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Trafficking - Example

• Association of transport vesicles with molecular
  motors and microtubules is controlled by
  phosphorylation state
• Phosphorylation of kinesin-1 enhances binding to
  sodium channel vesicles
• Dephosphorylation of kinesin-1 releases sodium
  channel vesicles
• Phosphatases enriched in Node of Ranvier
• Vesicle is trapped in Node of Ranvier