Intracellular Compartments and Protein Sorting

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Chapter 12

• Intracellular Compartments and Protein Sorting

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Intracellular compartments
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Not all cells are the same
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• Evolutionary origins of organelles

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Topological relationships of organelles can be
interpreted in terms of evolutionary origins
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Sidebar Construction of organelles

• Most membrane organelles cannot be constructed
  from scratch-they need the organelles to be
  present
• During division, daughter cells inherit portions
  of parent cell organelles
• Epigenetic inheritance

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• Topological relationship between compartments
  (red)
• Lumen of any of these compartments can
  communicate via transport vesicles

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Vesicle budding and fusion during vesicular
transport

• Orientation applies both to the lipid bilayer and
  transmembrane proteins

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Roadmap of protein traffic

• 3 modes of movement
• Gated traffic
• via nuclear pores
• Transmembrane transport
• membrane bound protein translocators
• Vesicular transport
• Movement between topologically related
  compartments
• Directed by signal sequences

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Terminal
Intrinsic

• Signal sequences and signal patches direct
  proteins to the correct cellular address

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• Positively charged- red
• Negatively charged-green
• Hydrophobic-white
• Hydroxylated-blue

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Roadmap of protein traffic

• 3 modes of movement
• Gated traffic
• via nuclear pores
• Transmembrane transport
• membrane bound protein translocators
• Vesicular transport
• Movement between topologically related
  compartments
• Directed by signal sequences

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Transport between the nucleus and the cytoplasm
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50 different Nucleoporins
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Gated diffusion paths through nuclear pore

• 9nm functional pore size for diffusion (lt5000Da)
• 26nm functional pore size for active transport

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Role of signal sequences in nuclear
localization SV40 virus T-antigen

• Normal protein located in nucleus
• Mutant protein located in cytoplasm

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Colloidal gold spheres coated with signal
sequence for nuclear import
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Import into nucleus requires additional
proteins 1. Nuclear import receptors (NIRs)

• Different cargo proteins contain different
  signals and bind to different NIRs

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FG repeats

• FG

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Import into nucleus requires additional
proteins 2. Ran transporter proteins

• Energy for import provided by Ran
• Located on both sides of nuclear envelope
• GTPase activity
• Localization depends upon Ran-GAP (import) and
  Ran GEF (export)

Ran GAP- Ran GTPase activating protein Ran GEF-
Ran guanine exchange factor
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Import into nucleus requires additional
proteins NIRs and Ran work together
Import Export
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Import receptor Ran GTP/GDP and cargo exchange
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• Nuclear envelope breaks down during mitosis
• Reassembles during telophase
• Lamins

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The breakdown and reformation of the nuclear
envelope during mitosis
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Roadmap of protein traffic

• 3 modes of movement
• Gated traffic
• via nuclear pores
• Transmembrane transport
• membrane bound protein translocators
• Vesicular transport
• Movement between topologically related
  compartments
• Directed by signal sequences

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Trafficking of proteins via transmembrane
transport

• Import into mitochondria and chloroplasts
• Import in ER
• Co-translational translocation
• Post-translational translocation
• Transmembrane proteins

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Roadmap of protein traffic

• 3 modes of movement
• Gated traffic
• via nuclear pores
• Transmembrane transport
• membrane bound protein translocators
• Vesicular transport
• Movement between topologically related
  compartments
• Directed by signal sequences

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• Positively charged- red
• Negatively charged-green
• Hydrophobic-white
• Hydroxylated-blue

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Import receptor

• Proteins imported in mitochondria and
  chloroplasts cross more than one membrane
• Signal sequence

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Mitochondrial translocators

• TOM
• Translocator of the Outer Membrane
• TIM
• Translocator of the Inner Membrane
• OXA
• Inner membrane

• Do proteins cross membranes simultaneously or
  sequentially?

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• Proteins transiently span both membranes during
  translocation into matrix

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Import into matrix

• Energy for translocation provided by ATP and H
  ion gradient
• Some proteins contain multiple signal sequences

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The role of energy in the import of proteins into
mitochondrial matrix space
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• Import into inter-membrane space
• A. Stop transfer sequence
• B. Two signal sequences
• C. Release of peptide into the inter-membrane
  space

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Import into inner membrane

• D. Formation of mitochondrial transmembrane
  proteins

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Translocation of precursor proteins into
thylakoid space of chloroplasts Part 1
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Translocation of precursor proteins into
thylakoid space of chloroplasts Part 2
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Endoplasmic reticulum
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• Smooth ER
• Membrane synthesis
• Vesicle formation

• Rough ER
• Protein synthesis and trafficking

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• Preparation of ER

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• Positively charged- red
• Negatively charged-green
• Hydrophobic-white
• Hydroxylated-blue

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Transport of protein into ER

• A. Co-translational translocation
• B. Post-translational translocation

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Co-translational translocation The signal
hypothesis

• Signal Sequences discovered in ER

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Signal Recognition Particle
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Signal recognition particle (SRP)
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Common pool of ribosomal subunits
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Transport of protein into ER

• A. Co-translational translocation
• B. Post-translational translocation

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Translocation of a soluble protein
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Single-pass transmembrane protein

• Hydrophobic region in membrane
• How does it stop translocation?

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• Integral membrane protein 1.

• Stop transfer sequence
• Amino terminal is inside the lumen (equivalent to
  extracellular)
• Carboxy terminal in cytosol

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Vesicle budding and fusion during vesicular
transport

• Orientation applies both to the lipid bilayer and
  transmembrane proteins

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• Integral membrane protein 2.

• Internal start-transfer sequence-allows amino
  terminus to remain in the cytosol. Carboxy
  terminal in lumen (equivalent to extracellular)
• No peptidase signal

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• Integral membrane protein 3.

Opposite orientation of internal signal sequence
allows amino terminus to remain in ER lumen
without a stop-transfer sequence
A
B
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Several ways that a protein can associate with a
membrane

• Single pass a-helix (1)
• Multi pass a-helix (2)
• b-barrel (3)
• Amphipathic a-helix (4)
• Lipid anchor (5)
• GPI anchor (6)
• Non-covalent association with membrane integral
  proteins 78)

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• Integration of double pass membrane protein
• Requires a start-transfer and a stop-transfer
  signal without a peptidase signal

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• Multiple pairs of start-transfer and
  stop-transfer signal sequences results in
  multi-pass membrane protein

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N-linked Glycosylation

• Oligosaccharide linked to asparagine in ER lumen
  (N-linked)
• Asp-X(not proline)-Ser/Thr
• Core region of oligosaccharide shaded

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Glycosylation

• Protein glycosylation in the ER occurs during
  translocation
• Oligosaccharide transferase
• Dolichol

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Why is glycosylation so common in ER

• Marks proteins for folding by chaperones

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Steps in creation of a functional protein
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Role of N-linked Glycosylation in ER protein
folding

• ER chaperone Calnexin
• Glucose added to cause binding to calnexin
• Protein retained in ER until fixed.

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Export and degradation of misfolded proteins

• Proteins are
• De-glycosylated
• Ubiquitinated
• Degraded in proteasomes

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Roadmap of protein traffic

• 3 modes of movement
• Gated traffic
• via nuclear pores
• Transmembrane transport
• membrane bound protein translocators
• Vesicular transport
• Movement between topologically related
  compartments
• Directed by signal sequences