THE DYNAMICS OF GOLGI PROTEIN TRAFFIC VISUALIZED IN LIVING YEAST-CELLS

We describe for the first time the visualization of Gels membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. La te and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutat ions on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and s ed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early ciste rnae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying th at it cycles through the ER. Electron microscopy shows that Golgi memb ranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and sati sfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, eit her within the Golgi complex or from there to the ER, as anterograde c argo advances.