MEMBRANE TRAFFICKING IN MAMMALIAN CELLS

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DESCRIPTION: In preliminary studies, the P.I. has provided evidence that Golgi and endosome membrane tubulation may be mediated by PLA2. This is based on an in vitro assay that reconstitutes Golgi tubulation using bovine brain cytosol. A factor of ~130 kD complex with a major polypeptide of 40 kD was isolated. Polyclonal antiserum can immuno-deplete the tubulation activity from BBC, indicating that this complex is indeed functional in vitro. That this factor may be PLA2 is provided by the finding that PLA2 inhibitors such as bromoenol lactone inhibit BFA induced Golgi tubulation in vivo and cytosol induced tubulation in vitro. A similar finding was obtained with endosomes. Furthermore, PLAP, an activator PLA2 activity stimulates retrograde transport in intact cells, and this effect is dependent on the presence of the 40 kD tubulation factor. Finally, the tubulation factor preparation contained phospholipase activity as assayed in vitro. The P.I. proposed three aims: (1) to molecularly characterize the 40 kD tubulation factor and study its enzymology, tissue distribution and membrane association, (2) to study endosome tubulation and factors involved, (3) functional analysis of PLA2/ membrane tubulation in membrane trafficking.