THE INTRACELLULAR MOBILITY OF A VIRAL MEMBRANE GLYCOPROTEIN MEASURED BY CONFOCAL MICROSCOPE FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins . However, the application of FRAP to the study of intracellular membr ane proteins has been hampered by the lack of specific probes and thei r physical inaccessibility in the cytoplasm. We have measured the mobi lity of a model transmembrane protein, the temperature-sensitive vesic ular stomatitis viral membrane glycoprotein (ts-045-G), in transit fro m the endoplasmic reticulum (ER) to the Golgi complex. ts-045-G accumu lates in the ER at nonpermissive temperature (39.5 degrees C) and is t ransported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab frag ments against a cytoplasmic epitope of ts-045-G (rh-P5D4-Fabs) were mi croinjected into cells to visualize the intracellular viral membrane p rotein and to determine its mobility by FRAP with a confocal microscop e. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-045-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-inje cted cells were bleached either following release of labeled ts-045-G from the ER or upon its accumulation at 20 degrees C in the trans-Golg i network (TGN). Zn contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to bet a-COP in vivo. The diffusion constant of mobile ts-045-G under all con ditions was similar to 10x10(-10) cm(2)/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane pr otein and provide the first evidence that such a protein is highly mob ile.