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Protein secretion/trafficking
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Compartmentalization
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Cells use membranes to isolate and concentrate specific
biochemical reactions inside the cell. A classic example of such compartmentalization
occurs in the secretory pathway where specific carbohydrate modifcations
of proteins are restricted to subcellular membrane organelles (e.g., endoplasmic
reticulum or the golgi).
Such compartmentalization requires the movement, or trafficking,
of proteins with a high degree of specificity. The secretory pathway is
able to recognize only those proteins destined for secretion or membrane
organelle residence and target those proteins to their final destination
using a combination of protein translocation (movement of proteins across
the lipid bilayer) and vesicular transport.
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Polarized Secretion
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Two examples help us understand why proper targeting of proteins is so
critical. Epithelial cells in the gut use the Na/Glucose symporter to
move glucose against its concentration gradient. This process has to occur
on the cell surface facing the lumen of the gut (the apical surface of
the cell), thus the Na/Glucose symporter must be secreted in a polarized
fashion to the apical surface of the plasma membrane.
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Migrating cells, such as those we observed after
wound
healing, form polarized cell adhesions (sticky stuff) at their
leading edge. In the movie below, GFP has been fused to a integral membrane
protein that allows cells to stick to the coverslip. Through changes in
the cell's cytoskeletal contractile forces mediate the movement of the cell
in the direction of the open wound. |
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Protein Translocation
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Proteins enter the secretory pathway via the
process of translocation. Unfolded proteins are threaded through a gated
type channel in the endoplasmic reticiculum (ER) known as the translocon
(Sec61 complex). Protein translocation occurs in discreted steps and is
robust. As observed in the trafficking of a viral protein fused to GFP in
the movie below, proteins move from the ER in vesicles, populate the golgi
and then are moved via vesicles to the plasma membrane. |
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Hirschberg et. al., 1998. ts045VSVG-GFP.
After shift to 32oC, the VSV-GFP is observed to traffick from the ER to
the golgi and the golgi to the plasma membrane. |
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Translocation of proteins into the ER is maintained
through G-proteins that act both as "switches" and "timers"
to ensure the proper order of steps and the recycling of complexes. In this
way, the secretory system is kept from being saturated by an excess of secreted
proteins. |
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Vesicular Transport
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Once proteins have entered the secretory pathway
through translocation, it must be properly targeted to its final cellular
destination (i.e., plasma membrane, lysosome, secretory vesicle, etc.).
At the same time, membrane organelles must maintain their "identity"
by retaining their resident proteins (e.g., BiP must stay in the ER). Secretory
protein targeting is carried out by the process of vescicular transport.
Trafficking of proteins out of the ER is termed anterograde transport and
the return of proteins from the golgi to the ER is termed retrograde transport.
Vesicular transport consists of budding, docking and fusion steps. |
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FRAP: Fluorescence Recovery After Photobleaching
of this lipid anchored GFP protein allows the observation of discrete vesicular
transport events. |
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Nicholes et. al., 2001 JCB. COS cells expressing
GPI-GFP. |
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