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J. Cell Biol.,
Volume 143, Number 6, December 14, 1998 1505-1521
* Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0308; During microtubule depolymerization, the
central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such
scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11-amino acid
epitope, VSV-G (VSV), and the trans/TGN enzyme
Cell
Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69012 Heidelberg, Germany; and § Department of Anatomy, Miyazaki Medical College, Miyazaki, 889-1692 Japan
1,4-galactosyltransferase (GalT) fused to GFP. After
nocodazole addition, time-lapse microscopy of GalNAc-T2-GFP and GalT-GFP revealed that scattered
Golgi elements appeared abruptly and that no Golgi
fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for
tens of minutes. During the entire process of dispersal,
immunogold labeling for GalNAc-T2-VSV and GalT
showed that these were continuously concentrated over
stacked Golgi cisternae and tubulovesicular Golgi
structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In
fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident
proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through
the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export.
Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2-VSV in the ER. Few to no
peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion,
we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced
Golgi scattering observed in interphase cells.
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