MORPHOLOGICAL ANALYSIS OF THE TRANSFER OF VSV TS-045 G-GLYCOPROTEIN FROM THE ENDOPLASMIC-RETICULUM TO THE INTERMEDIATE COMPARTMENT IN VERO CELLS

Vero cells were infected with the ts-045 strain of vesicular stomatiti s virus, and the cells were incubated at 39 degrees C to accumulate th e mutant G glycoprotein in the ER as a misfolded aggregate. Cyclohexim ide was added to the culture medium 3.5 h after infection to prevent f urther protein synthesis, and the temperature was lowered to 10, 15, o r 31 degrees C. At these temperatures, the mutant G glycoprotein corre ctly folds and oligomerizes; Immunofluorescence light microscopy showe d that the G glycoprotein was exported to the Golgi complex at 31 degr ees C and to the intermediate compartment (IC) at 15 degrees C, but no export was observed at 10 degrees C. However, incubations at 10 degre es C followed by shift to 15 or 31 degrees C resulted in the normal tr ansfer of the glycoprotein to the IC and the Golgi respectively, Immun oelectron microscopical analysis confirmed all these results, but show ed also that the glycoprotein was frequently clustered in the ER at 10 degrees C. Conventional electron microscopy showed that the morpholog y of the ER, IC, and Golgi complex remained essentially unchanged at a ll temperatures. The only significant difference detectable in cells i ncubated at 10 degrees C was the increased number of partially coated ER protrusions, longer than those detected at higher temperatures. The se results demonstrate that the transport toward the Gels complex of G glycoprotein can be arrested at a step preceding the entry into the I C, thus suggesting that ER and IC are separate stations in the exocyti c pathway. (C) 1996 Academic Press, Inc.