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 has been proposed to require the function of heterotrimeric G proteins. To begin to identify the basic elements of a potential signaling pathway involved in vesicle assembly, we have examined whether protein kinase C (PKC) is required for ER to Golgi transport. Calphostin C, a specific inhibitor of the highly conserved cysteine-rich C6H2 motif present in the regulatory domain of PKC was found to be a potent inhibitor of export of VSV-G and vesicle budding from the ER in vivo and in vitro (IC50 approximately 60 nM). In contrast, the diacylglycerol analog phorbol 12-myristate 13-acetate, which activates PKC, enhanced the migration of VSV-G from the ER to pre-Golgi intermediates. Neither reagent had detectable effects on the oligomerization of VSV-G prior to export nor perturbed transport of protein between compartments of the Golgi stack. In contrast to the striking effects of calphostin C, reagents that inhibit the function of the catalytic domain of PKC (including the general kinase inhibitor staurosporine, as well as the more specific inhibitors H-7, H-8, pseudosubstrate inhibitor, or chelerythrine) did not inhibit export from the ER. Export was also insensitive to down-regulation of various PKC isoforms. These results suggest that a novel protein containing the conserved C6H2 motif may serve as a potential link in a signaling pathway regulating vesicle budding from the ER.
Fabbri
M
M
Department of Cell and Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Bannykh
S
S
Balch
W E
WE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002451:Cell Compartmentation; D017124:Conserved Sequence; D006224:Cricetinae; D004075:Diglycerides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D009281:Naphthalenes; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D009281:Naphthalenes; D011083:Polycyclic Compounds; D011955:Receptors, Drug; D014759:Viral Envelope Proteins; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate