Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering

During microtubule depolymerization, the central, juxtanuclear Golgi appara tus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracki ng of Golgi units or if peripheral Golgi elements reform through a novel re cycling pathway. To mark the Golgi in HeLa cells, we stably expressed the G olgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an Il-amino acid epitope, VSV-G ( VSV), and the trans/TGN enzyme beta 1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2-GFP a nd GalT-GFP revealed that scattered Golgi elements appeared abruptly and th at no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fl uorescence intensity for tens of minutes. During the entire process of disp ersal, immunogold labeling for GalNAc-T2-VSV and GalT showed that these wer e continuously concentrated over stacked Golgi cisternae and tubulovesicula r Golgi Golgi stacks reform rapidly at scattered sites. In fluorescence rec overy after photobleaching over a narrow (FRAP) or wide area (FRAP-W) exper iments, peripheral Golgi stacks continuously exchanged resident proteins wi th each other through what appeared to be an ER intermediate. That Golgi en zymes cycle through the ER was confirmed by microinjecting the dominant-neg ative mutant of Sari (Sar1p(dn)) blocking ER export. Sar1p(dn) was either m icroinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumula tion of GalNAc-T2-VSV in the ER. Few to no peripheral Golgi elements were s een in the nocodazole-treated cells microinjected with Sar1p(dn). In conclu sion, we have shown that Golgi-resident glycosylation enzymes recycle throu gh the ER and that this novel pathway is the likely explanation for the noc odazole-induced Golgi scattering observed in interphase cells.