Protein trafficking'

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Protein trafficking. Lecture 10, Fall 2004
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Typical eucaryote contains multiple internal
membrane compartments.
A typical procaryote lacks internal membrane
compartments.
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Most newly synthesized proteins begin synthesis
in the cytosol and their destinies depend on
sorting signals encoded in the amino acid
sequence of the protein. Proteins synthesized in
the cytosol and lacking sorting signals remain in
the cytosol. A small number of proteins do not
begin synthesis in the cytosol. These proteins
are found in mitochondria and chloroplasts. They
are encoded by DNA present in these organelles
and are synthesized by ribosomes residing in the
lumen of these organelles.
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Roadmap of protein traffic.
The genesis and function of internal compartments
depends on the appropriate targeting of proteins
Some of the green route illustrated.
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A more complicated sorting signal consists of a
signal patch generated by the tertiary
structure of the protein.
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Biosynthetic-secretory pathway.
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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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• Functions of the ER.
• Starting point for newly synthesized proteins
  destined for Golgi, Endosome, Lysosomes,
  Secretory vesicles, and the Plasma membrane (see
  below).
• Establishes orientation of proteins in the
  membrane.
• Site of phospholipid and cholesterol synthesis.
• Initiation site for N-linked glycosylation of
  proteins.

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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 fusing to the plasma
membrane, the arrow head would be projecting into
the extracellular space.
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Vesicular transport is performed by various
proteins that must do 3 things 1. Form the
vesicle with the correct cargo. 2. Target the
vesicle to the correct destination. 3. Fuse the
vesicle to the target membrane.
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Three coat proteins drive vesicle formation at
various locations in the cell.
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Clathrin associates via adaptins with receptors
in the donor membrane. The receptors bind
specific cargo. The clathrin assembles into a
cage that encapsulates a region of membrane.
Then dynamin causes the membrane to pinch off
forming a vesicle.
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TEM detection of clathrin cages forming at the
plasma membrane.
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Insert figure 13-7 to show assembly
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• Clathrin-dependent vesicle formation requires
  energy.
• GTP hydrolysis by dynamin accompanies pinching
  off.
• ATP hydrolysis by chaperone proteins (not shown)
  accompanies the dismantling of the clathrin coat
  - the chaperone protein in this case is changing
  the folding of the clathrin so the molecules
  dissociate from each other.

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COPII vesicle formation is mediated by a
monomeric GTPase. A GEF in the donor membrane
interacts with the GTPase, Sar1, causing GDP/GTP
exchange. Sar1-GTP extends a fatty acid tail
that inserts into the membrane. COPII assembles
on the Sar1 to form a vesicle.
COPI vesicle formation involves a protein called
ARF that is analogous to Sar1.
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Uncoating of the COPI and COPII vesicles occurs
when the G-protein (ARF or Sar1) hydrolyzes GTP
and retracts that fatty acid tail. This may not
require a GAP but is instead dictated by the rate
of hydrolysis intrinsic to the G-protein.
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Targeting of the vesicles is achieved by
complementary sets of v-SNAREs and t-SNAREs.
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Conformational changes in the v-SNARE/t-SNARE
complex appear to drive membrane fusion without
ATP or GTP hydrolysis.
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After membrane fusion, ATP hydrolysis is used to
pry apart the v-SNARE/t-SNARE complex.