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© The Rockefeller University Press, 0021-9525/1998//1505 $5.00
The Journal of Cell Biology, Volume 143, Number 6, , 1998 1505-1521

Article

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering



Brian Storrie*, Jamie White{ddagger}, Sabine Röttger{ddagger}, Ernst H.K. Stelzer{ddagger}, Tatsuo Suganuma§, and Tommy Nilsson{ddagger}

* Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0308; {ddagger} Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69012 Heidelberg, Germany; and § Department of Anatomy, Miyazaki Medical College, Miyazaki, 889-1692 Japan

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 β1,4-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.

Key Words: Golgi apparatus • endoplasmic reticulum • Sar1p • protein cycling • nocodazole

Abbreviations used in this paper: CHX, cycloheximide; FRAP, fluorescence recovery after photobleaching over a narrow area; FRAP-W, fluorescence recover after photobleaching over a wide area; GalNAc-T2, N-acetylgalactosaminyltransferase-2; GalT, β1,4-galactosyltransferase; GFP, green fluorescent protein; PDI, protein disulfide isomerase; VSV-G, vesicular stomatitis virus G protein.

This project was a joint effort between the laboratories of Brian Storrie at Virginia Tech and those of Ernst Stelzer and Tommy Nilsson at EMBL-Heidelberg. During this project, Jamie White and Sabine Röttger were predoctoral students at EMBL-Heidelberg, and the work of Jamie White was jointly sponsored by Ernst Stelzer and Tommy Nilsson and that of Sabine Röttger by Tommy Nilsson. At Virginia Tech-Blacksburg, we would like to express our appreciation to Jeffrey Bocock, Sarah Buss, Karen Capen, and Nolan Ko for quantification of various morphometric measurements and fluorescence intensities. At EMBL-Heidelberg, we would like to express our appreciation to the CCC developers: Nick Salmon, Alfons Riedinger, Georg Ritter, Stephan Albrecht, Thomas Stephany, and Reiner Stricker, to Ann Atzberger for FACS®, to Anja Habermann for cryosectioning, and to Joel Lanoix for his help on Sar1pdn purification. We gratefully acknowledge the critical comments on the manuscript of Rich Walker and Brenda Shirley at Virginia Tech and of Joachim Füllekrug, Joel Lanoix, and Heidi McBride at EMBL-Heidelberg.

The first two authors contributed equally to the paper.

Requests for plasmids or cell lines from within North America should be directed to Brian Storrie and from within the European Union to Tommy Nilsson.


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