Protein Trafficking continued

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Protein Trafficking continued Lecture 12, Fall
2003
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Overview of the biosynthetic pathway of many
membrane proteins and secreted proteins and of
endocytosis.
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Rough ERs rough appearance is due to
polyribosomes.
ER lumen is one continuous space that merges with
the perinuclear space.
12-38
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• A signal sequence of approximately 20 amino acids
  and rich with hydrophobic amino acids is often
  located at the N-terminus.
• Since the ribosome masks about 30 amino acids,
  the signal sequence isnt fully exposed until the
  nascent polypeptide is about 50 amino acids long.
• SRP-ribosome attaches to SRP receptor and then
  docks on a protein translocator.
• SRP and receptor dissociate.
• Translation and translocation proceed in unison -
  co-translational transport.
• The energy for transport is provided by the
  translation process - as the polypeptide grows,
  it is pushed through the protein translocator.

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The signal sequences of secreted and soluble
proteins are cleaved by a signal peptidase. In
the literature, the signal sequence of secreted
proteins is often called a leader peptide.
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Co-translational transport must be able to
generate a diverse array of configurations.
For both single-pass and multipass transmembrane
proteins, some types will have the N-terminus
projecting into the cytosol and others will have
the C-terminus projecting into the cytosol.
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Orientation is established during synthesis by
start-transfer and stop-transfer sequences.
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These two examples have an internal
start-transfer sequence. In the top case, the
orientation is such that translation by the
ribosome pushes the growing chain through the
translocator. In the bottom case, the
orientation is such that ribosome separates from
the protein translocator and chain growth occurs
in the cytosol.
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In this case, a start transfer sequence followed
by a stop transfer sequence yields a 2-pass
transmembrane protein with the N and C-terminus
in the cytosol.
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Things can get pretty complicated!
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The orientation of a multipass transmembrane
protein depends two things 1. The direction in
which the hydrophobic domain closest to the
N-terminus inserts into the protein
translocator. 2. The order of hydrophobic domains
following the hydrophobic domain that initiated
co-translational transport. Whether a
hydrophobic domain functions as a start transfer
or stop transfer sequence depends on where it is
relative to preceding hydrophobic domains.
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• Activities that occur in the ER.
• Chaperons including BiP mediate correct
  folding.
• Protein disulfide isomerase mediates correct
  formation of disulfide bonds (note that disulfide
  bonds are normally absent from proteins found in
  the cytosol or nucleus, but present in proteins
  exposed outside the cell)
• Glycosylation of specific asparagine side chains
  - N-linked glycosylation (since this occurs in
  the lumen of the ER, the polysaccharides end up
  on the outside of the cell)

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Transport from the ER to the Golgi and beyond
occurs by vesicular transport.
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Vesicular transport maintains a specific
topological relationship between oraganelles and
the outside of the cell inside the organelles
connected by vesicular transport equals the
outside of the cell.
If this was a vesicle fusion to the plasma
membrane, the arrow head would be projecting into
the extracellular space.
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Vesicle formation. Clathrin or COP proteins
associate with membrane proteins and form the
vesicle. Cargo is selected by receptors that
interact with Clathrin or COP proteins. The
cargo destined for places other than the plasma
membrane will have a signal sequence that binds
the receptors. Vesicle targeting depends on
SNARES. v-SNARES associated with the vesicles
direct the vesicles to a destination containing a
matching t-SNARE. Vesicle fusion. The
v-SNARE/t-SNARE complex undergoes a
conformational change that forces the membranes
to fuse.
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Transport from the ER through the Golgi Apparatus
- continuation of the biosynthetic-secretory
pathway.
Properly folded proteins are loaded into COPII
transport vesicles, while unfolded proteins
remain associated with chaperones in the ER until
folding is complete.
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The Golgi Apparatus has two major functions 1.
Modifies the N-linked oligosaccharides and adds
O-linked oligosaccharides (O-link is to serine
or threonine). 2. Sorts proteins so that when
they exit the trans Golgi network, they are
delivered to the correct destination.
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The acid hydrolases in the lysosome are sorted in
the TGN based on the chemical marker mannose
6-phosphate.
Hydrolases are transported to the late endosome
which later matures into a lysosome.
Acidic pH causes hydrolase to dissociate from the
receptor.
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Nuclear transport transport folded proteins
through nuclear pores, nuclear import and export
receptor, Ran, Ran-GEF, Ran-GAP Mitochondrial
import transport unfolded proteins from cytosol,
chaperons, TOM and TIM ER cotranslational
transport, SRP, SRP receptor, Protein
translocator, membrane orientation Green routes
COP, clathrin, tSNARE, vSNARE, membrane
orientation