EXPORT OF PROTEIN FROM THE ENDOPLASMIC-RETICULUM IS REGULATED BY A DIACYLGLYCEROL PHORBOL ESTER BINDING-PROTEIN

The export of vesicular stomatitis virus glycoprotein (VSV-G) from the endoplasmic reticulum (ER) involves sorting and concentration, and ha s been proposed to require the function of heterotrimeric G proteins. To begin to identify the basic elements of a potential signaling pathw ay involved in vesicle assembly, we have examined whether protein kina se C (PKC) is required for ER to Golgi transport. Calphostin C, a spec ific inhibitor of the highly conserved cysteine-rich C6H2 motif presen t in the regulatory domain of PKC was found to be a potent inhibitor o f export of VSV-G and vesicle budding from the ER in vivo and in vitro (IC50 similar to 60 nM). In contrast, the diacylglycerol analog phorb ol 12-myristate 13-acetate, which activates PKC, enhanced the migratio n of VSV-G from the ER to pre-Golgi intermediates. Neither reagent had detectable effects on the oligomerization of VSV-G prior to export no r perturbed transport of protein between compartments of the Golgi sta ck. In contrast to the striking effects of calphostin C, reagents that inhibit the function of the catalytic domain of PKC (including the ge neral kinase inhibitor staurosporine, as well as the more specific inh ibitors H-7, H-8, pseudosubstrate inhibitor, or chelerythrine) did not inhibit export from the ER. Export was also insensitive to down-regul ation of various PKC isoforms. These results suggest that a novel prot ein containing the conserved C6H2 motif may serve as a potential link in a signaling pathway regulating vesicle budding from the ER.