GRASP55 Regulates Golgi Ribbon Formation

Abstract

Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression. INTRODUCTIONThe structural diversity of the Golgi apparatus among eukaryotes suggests two primary modes of organization. Cis-, medial-, and trans-Golgi cisternae, although unconnected in budding yeast, are typically present as membrane stacks. 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 Gol...

Membrane adhesion dictates Golgi stacking and cisternal morphology

“…An appealing mechanism for intercisternal adhesion has been proposed for the GRASP proteins based on X-ray crystallography and biochemistry that involves PSD-95, Dlg1, Zo-1 domain-dependent homo-oligomerization in trans (13-15). In recent years, with the advent of RNAi-based technologies, knock-down studies have broadly confirmed a role for GRASP proteins and Golgins in controlling Golgi morphology but have not agreed with each other on many notable details, leaving the field in a somewhat confused and conflictory state (10,(15)(16)(17)(18)(19)(20). In the simpler case of Drosophila, dsRNA-mediated depletion of dGRASP results in ∼30% loss of Golgi stacks, whereas double depletion of dGRASP and dGM130 was shown to unstack the Golgi, as examined by EM (21).…”

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

“…In addition to Golgi stacking, GRASP proteins have been implicated in lateral fusion of cisternae to form ribbon-like Golgi structures in mammalian cells (22,23) and in unconventional secretion pathways (24 -26). The membrane-tethering activity appears to be critical for the functions of GRASP proteins.…”

GRASP65 and GRASP55 Sequentially Promote the Transport of C-terminal Valine-bearing Cargos to and through the Golgi Complex

“…This activity arose as the result of the tethering functions displayed by GRASP65 and GRASP55, through their interactions with their partner proteins GM130 and golgin-45, respectively (2)(3)(4). Several other studies have shown more recently that the GRASPs are involved in the maintenance of the structure of the Golgi ribbon in mammal cells during interphase, in controlling the fragmentation of the Golgi complex at the onset of mitosis (5)(6)(7)(8), in establishing cell polarity in migrating cells (9), and in the consumption of COPII vesicles and the formation of the cis-Golgi in yeast (10).…”

“…Indeed, a direct role for GRASPs in cargo transport has so far been established only for the unconventional secretion routes in Dictyostilium and Drosophila (11,12), whereas they have been shown not to be directly involved in the trafficking of commonly studied reporter cargo proteins along the "conventional" secretory pathway (e.g. the temperature-sensitive (ts-045) mutant of the G protein of vesicular stomatitis virus (VSVG) 2 ) and secretory horseradish peroxidase (5,6,13,14). GRASPs can engage different types of interactions including the ones mediated by their PDZ domains, through which the GRASPs cannot only homodimerize, thus participating in cisternal stacking (15) but can also bind the C-terminal valine motifs (C-TVM) of membrane proteins such as protransforming growth factor ␣ and p24a (16,17).…”