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

摘要: During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due a fragmentation process and subsequent outward tracking of units or if elements reform through novel recycling pathway. To mark in HeLa cells, we stably expressed stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused green fluorescent protein (GFP) an 11-amino acid epitope, VSV-G (VSV), trans/TGN beta1,4-galactosyltransferase (GalT) GFP. After nocodazole addition, time-lapse microscopy GalNAc-T2-GFP GalT-GFP revealed that scattered appeared abruptly no fragments tracked from compact, complex. Once formed, structures were relatively stable fluorescence intensity for tens minutes. entire dispersal, immunogold labeling GalNAc-T2-VSV GalT showed these continuously concentrated over stacked cisternae tubulovesicular similar untreated suggesting polarized stacks rapidly at In recovery after photobleaching narrow (FRAP) wide area (FRAP-W) experiments, exchanged resident proteins with each other what be ER intermediate. That enzymes cycle was confirmed by microinjecting dominant-negative mutant Sar1 (Sar1pdn) blocking export. Sar1pdn either microinjected into nocodazole-treated cells presence synthesis inhibitors. both cases, this caused gradual accumulation ER. Few seen Sar1pdn. conclusion, shown Golgi-resident glycosylation recycle pathway likely explanation nocodazole-induced observed interphase cells.