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5 Analysis of Secretory Protein Trafficking Within Living Cells

We used the thermoreversible folding mutant, tsO45 vesicular stomatis viral G protein, fused to green fluorescent protein (VSVG-GFP) as reporter to study the dynamic properties of secretary traffic in living cells. The tsO45 VSVG protein misfolds and is retained in the ER at 4OC, but upon temperature shift to 32C moves as a synchronous population to the Golgi complex before being transported to the plasma membrane. These properties are preserved after tagging VSVG at its cytoplasmic tail with GFP. To study the morphological and kinetic properties of VSVG-GFP transport through various compartments of the secretary pathway, we used digital fluorescent imaging techniques to analyze time-lapse confocal sequences of VSVG-GFP transport from the ER to the plasma membrane. With this approach we determined kinetic parameters for movement of a given population of VSVG-GFP through the secretory pathway, including the time for VSVG-GFP to transit Golgi stacks, its rate of Golgi egress and conditions that affect export of VSVG-GFP out of the Golgi complex. Because the number of GFP molecules per VSVG-GFP is fixed at one per chimera, we could also estimate the number of GFP chimeras being imaged in any single cell by comparing total cellular GFP fluorescence with a known concentration of GFP. This allowed us to determine VSVG-GFP flux (i.e. molecules per sec) in and out of different compartments. The processes of budding, translocation and fusion of pre- and post-Golgi transport intermediates were also examined. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for delivery of VSVG-GFP between different stations of the secretory pathway.

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