Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells

Quantitative time-lapse imaging data of single cells expressing the transme mbrane protein, vesicular stomatitis virus ts045 G protein fused to green f luorescent protein (VSVG-GFP), were used for kinetic modeling of protein tr affic through the various compartments of the secretory pathway. A series o f first order rate laws was sufficient to accurately describe VSVG-GFP tran sport, and provided compartment residence times and rate constants for tran sport 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 V SVG-GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membr ane transport it was 3.0% per min, and for transport from the plasma membra ne 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), s ecretory 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 structure s, rather than small vesicles, were found to be the primary vehicles for Go lgi to plasma membrane transport of VSVG-GFP, These structures budded as en tire 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 m in. In addition, they fused with the plasma membrane without intersecting o ther membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for co nveying large quantities of protein cargo from the Golgi complex directly t o the plasma membrane.