A direct role for GRASP65 as a mitotically regulated Golgi stacking factor

Abstract

contributed equally to this work Cell-free assays that mimic the disassembly and reassembly cycle of the Golgi apparatus during mitosis implicated GRASP65 as a mitotically regulated stacking factor. We now present evidence that GRASP65 is directly involved in stacking Golgi cisternae. GRASP65 is the major phosphorylation target in rat liver Golgi membranes of two mitotic kinases, cdc2±cyclin B and polo-like kinases, which alone will unstack Golgi membranes, generating single cisternae. Mitotic cells microinjected with antibodies to GRASP65 fail to form proper Golgi stacks after cell division. Beads coated with GRASP65 homodimers form extensive aggregates consistent with the formation of trans oligomers. These can be disaggregated using puri®ed cdc2±cyclin B1 and polo-like kinases, and re-aggregated after dephosphorylation of GRASP65. Together, these data demonstrate that GRASP65 has the properties required to bind surfaces together in a mitotically regulated manner.

GRASP55 Regulates Golgi Ribbon Formation

“…GRASP55-T 222,225 D was also defective in oligomer formation, supporting a model in which the homotypic fusion underlying Golgi ribbon formation depends on membrane cross-bridging by GRASP55 transoligomers. This model for the function of mammalian GRASP proteins largely agrees with previous work on Golgi assembly (Barr et al, 1997;Shorter et al, 1999;Wang et al, 2003Wang et al, , 2005, but its relevance to other described functions for GRASP55 and GRASP65 remains to be determined. GRASP55 binds and may facilitate transport of the transmembrane growth factor ␣ (Kuo et al, 2000), and both GRASP proteins bind members of the p24 protein family, thereby inhibiting p24 recycling to the ER (Barr et al, 2001).…”

“…There may also be a sequential relationship in the regulation of the two proteins at the onset of mitosis. Evidence suggests that GRASP55 is phosphorylated in late G2 before CDK1 activation, and, therefore, that its regulation occurs upstream of GRASP65 phosphorylation by CDK1 and CDK1-activated PLK1 (Elia et al, 2003a,b;Wang et al, 2003;Preisinger et al, 2005;Feinstein and Linstedt, 2007). If so, GRASP65 phosphorylation might be considered redundant with respect to unlinking the Golgi.…”

“…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).…”

“…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.…”

Endoplasmic reticulum architecture: structures in flux

“…Thus, the stacked arrangement of membranes, as observed with Golgi cisternae [55] or thylakoid membranes, could be caused by weak, transient interactions like the ones involved in OSER formation, rather than requiring a specific matrix or 'glue' for holding them together. Such transient interactions could also underlie junctions between the ER and other organelles and could be subject to regulation.…”