3 DISTINCT STEPS IN TRANSPORT OF VESICULAR STOMATITIS-VIRUS GLYCOPROTEIN FROM THE ER TO THE CELL-SURFACE IN-VIVO WITH DIFFERENTIAL SENSITIVITIES TO GTP-GAMMA-S

Microinjected GTP gamma S revealed three distinct steps in the exocyti c transport of the temperature sensitive glycoprotein of vesicular sto matitis virus (ts-O45-G) from the ER to the cell surface in intact Ver o cells. While COPII dependent export of ts-045-G from the ER is block ed in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with thi s transport step significantly. In contrast, transport to the Golgi co mplex is blocked by 50 mu M GTP gamma S, a dominant mutant of ARF1 (AR F1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sarla[H79G]p has no effect, Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated v esicular structures lacking ts-O45-G. Finally, transport of ts-045-G f rom the trans-Golgi network (TGN) to the cell surface is inhibited onl y by high concentrations of GTP gamma S (500 mu M). Interestingly, thi s step is only partially brefeldin A sensitive, and injected antibodie s against beta-COP and p200/myosin II, a TGN membrane associated prote in, have no effect, These data provide first strong in vivo evidence f or at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-045-G. COPI plays a rol e in subsequent transport to the Golgi complex, and a so far unidentif ied GTP gamma S sensitive coat appears to be involved in transport fro m the TGN to the cell surface.