GM130 and GRASP65-dependent lateral cisternal fusion allows uniform Golgi-enzyme distribution

Abstract

The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.

GRASP55 Regulates Golgi Ribbon Formation

“…Significantly, the Golgi ribbon was disrupted in cells that lacked GRASP55 staining. The observed phenotype was similar to that previously noted after GM130 or GRASP65 knockdown (Puthenveedu et al, 2006), and quantification using the same image analysis protocol indicated a similar degree of Golgi fragmentation ( Figure 1C). The analysis was then carried out for cells arrested at late G2 phase of the cell cycle when the ribbon undergoes MEKdependent unlinking (Feinstein and Linstedt, 2007).…”

“…In contrast, fluorescent Golgi objects in GRASP55-depleted cells, even when they seemed optically contiguous, failed to recover significantly from photobleaching. These results were quantified for multiple experiments ( Figure 2B), and representative movies are present in Supplemental Figure S1 and S2 movies.An earlier study found that disruption of the Golgi ribbon correlated with discontinuities in Golgi enzyme distribution and perturbed sialylation, resulting in an increase in nonsialylated proteins detectable on the cell surface (Puthenveedu et al, 2006). This was also the case for cells exhibiting an unlinked Golgi due to GRASP55 depletion.…”

“…Whereas many cell types contain numerous scattered stacks positioned adjacent to endoplasmic reticulum (ER) exit sites, known as ministacks, mammalian cells and those from related metazoans exhibit a pericentrosomal Golgi in which ministacks are laterally linked into a ribbon. The in vivo requirements for Golgi stack formation remain unknown; however, recent work has begun to identify components necessary for linking cisternal stacks into a contiguous Golgi ribbon.Depletion of the golgin Golgi matrix protein 130 kDa (GM130), or its binding partner Golgi reassembly protein (GRASP)65, by using RNA interference (RNAi) specifically disrupts formation of the Golgi ribbon (Puthenveedu et al, 2006). GM130 seems to recruit GRASP65 to Golgi membranes because Golgi localization of GRASP65 is lost upon GM130 knockdown, whereas GM130 remains Golgi localized in the absence of GRASP65 (Sutterlin et al, 2005;Puthenveedu et al, 2006).…”

“…A mechanism for Golgi ribbon formation by GRASP65 is suggested by the finding that two postsynaptic density 95/disc-large/zona occludens-like domains at the N terminus of GRASP65 form oligomers that, in the presence of cytosol, can cross-link beads (Wang et al, 2003(Wang et al, , 2005. Thus, the Golgi ribbon may be formed by GM130-dependent recruitment of GRASP65 to Golgi cisternal rims where GRASP65 transoligomer formation crossbridges adjacent cis-cisternae and possibly primes them for soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated membrane fusion (Puthenveedu et al, 2006).The lateral contacts that form the Golgi ribbon seem dynamic. The C-terminal binding protein/brefeldin A-ADPribosylated substrate (CtBP/BARS) disrupts the Golgi ribbon, possibly catalyzing membrane fission at contact sites (Weigert et al, 1999;Bonazzi et al, 2005).…”

“…This finding was supported by quantitative analysis of multiple experiments ( Figure 8B). Based on its similarity to GRASP65, it could be proposed that homo-oligomerization of GRASP55 cross-bridges membranes to form the Golgi ribbon, and this activity might be inhibited by phosphorylation (Wang et al, 2003(Wang et al, , 2005Puthenveedu et al, 2006;Feinstein and Linstedt, 2007). To test whether GRASP55 can interact with itself, purified GSTtagged GRASP55 immobilized on agarose beads was incubated with extracts prepared from HeLa cells transiently expressing myc-tagged GRASP55.…”