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

Abstract

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.

The Golgi Complex Is a Microtubule-organizing Organelle

“…As shown for WIF-B cells (Figure 4B), the Golgi remained essentially in a centrosomal location, although very small Golgi fragments also occurred throughout the cytoplasm and were probably rebuilt from the ER (Cole et al, 1996;Storrie et al, 1998). In these conditions, there was a dramatic drop in the amount of noncentrosomal MTs ( Figure 4B) compared with cells in which nocodazole treatment was performed at 37°C ( Figure 4A).…”

“…This means that our in vivo observations were likely not to result from the severing and release of centrosomal MTs (Baas and Joshi, 1992;Yu et al, 1993;Keating et al, 1997;Vorobjev et al, 1997) or even spontaneous cytoplasmic assembly, followed by treadmilling or migration en bloc Vorobjev et al, 1997;Yvon and Wadsworth, 1997;Tucker et al, 1998) until they were captured and stabilized by scattered Golgi elements. Because the reconstitution of mini-Golgi stacks at ER exit sites actively participates in the dispersion of the Golgi complex after nocodazole-mediated MT depolymerization (Cole et al, 1996;Storrie et al, 1998), the ER would also have been a likely candidate for stimulating noncentrosomal MT assembly. This possibility was clearly ruled out because the vast majority of newly assembled MTs followed the Golgi when the organelle was kept in a central location during MT depolymerization.…”

“…Conversely, the central localization of the Golgi apparatus involves cytoplasmic dynein (Corthésy-Theulaz et al, 1992;Fath et al, 1994;Burkhardt et al, 1997;Harada et al, 1998). It is also worth noting that, in addition to the kinesin-mediated disruption of the central Golgi complex during MT depolymerization, the dispersion of Golgi elements also relies on the reconstitution of mini Golgi stacks at endoplasmic reticulum (ER) exit sites (Cole et al, 1996;Storrie et al, 1998). Such data on the scattering and the reclustering of the Golgi complex have led to the view that Golgi membranes undergo an intimate relationship with MTs and especially with a population of nocodazole-resistant, stable MTs.…”

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

“…It is estimated that at least 5% of Golgi resident enzymes reside in the ER, at steady state (Pelletier et al, 2000). This pool constitutes recycled material originating from the Golgi apparatus (Cole et al, 1998;Storrie et al, 1998).…”

“…It is estimated that at least 5% of Golgi resident enzymes reside in the ER, at steady state (Pelletier et al, 2000). This pool constitutes recycled material originating from the Golgi apparatus (Cole et al, 1998;Storrie et al, 1998).The extent of ER recycling of Golgi resident membrane proteins is sufficient to allow for complete assembly of functional Golgi stacks. Thus, upon addition of nocodazole to depolymerize microtubules, the central and juxtanuclear Golgi apparatus relocates to the 100 or so peripheral ER exit sites scattered throughout the cell.…”