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*ALUMINUM FLUORIDE
*CYTOCHALASIN B
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© The Rockefeller University Press, 0021-9525/1998//1485 $5.00
The Journal of Cell Biology, Volume 143, Number 6, , 1998 1485-1503

Article

Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells



Koret Hirschberg, Chad M. Miller, Jan Ellenberg, John F. Presley, Eric D. Siggia{ddagger}, Robert D. Phair§, and Jennifer Lippincott-Schwartz

Cell Biology and Metabolism Branch, National Institutes of Health, National Institute of Child Health and Human Development, Bethesda, Maryland 20892; {ddagger} Center for Studies of Physics and Biology, Rockefeller University, New York, New York 10021; and § BioInformatics Services, Rockville, Maryland 20854

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments.

The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

Key Words: Golgi-to-plasma membrane traffic • GFP • secretion • kinetics • transport intermediates

Abbreviations used in this paper: cyto B, cytochaslin B; FWHM, full width half maximum; GFP, green fluorescent protein; PGC, post-Golgi carrier; ROI, regions(s) of interest; SIT, silicon-intensified target; VSVG– GFP, vesicular stomatitis virus G protein fused to GFP.

C. Miller is a Howard Hughes Medical Institute Scholar at the National Institutes of Health. K. Hirschberg is funded by the Human Frontiers Science Program.


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