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MRC Laboratory of Molecular Biology, Cambridge CB2 2QH,
United Kingdom
We describe for the first time the visualization of Golgi membranes
in living yeast cells, using green fluorescent protein (GFP) chimeras.
Late and early Golgi markers are present in distinct sets of scattered,
moving cisternae. The immediate effects of temperature-sensitive
mutations 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 sed5
cells, but not in sec14 cells. This is consistent with
retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to
early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p
itself moves rapidly to the endoplasmic reticulum (ER) in
sec12 cells, implying that it cycles through the ER.
Electron microscopy shows that Golgi membranes vesiculate in
sec18 cells within 10 min of a temperature shift. These
results emphasize the dynamic nature of Golgi cisternae and satisfy the
kinetic requirements of a cisternal maturation model in which all
resident proteins must undergo retrograde vesicular transport, either
within the Golgi complex or from there to the ER, as anterograde cargo
advances.
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