dGRASP Localization and Function in the Early Exocytic Pathway inDrosophilaS2 Cells

Abstract

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning:Arabidopsis h-Type Thioredoxins as a Case Study

“…It is interesting to notice that, for few of them, the N-terminal MYR alone without any evident second signal has been shown to target these proteins to intracellular membrane compartments with the ER/Golgi complex being the most frequent localization observed (Resh, 2006). For instance, this is the case for proteins such as eNos (Liu et al, 1997), GRASP (Kondylis et al, 2005), and the Arl family (Price et al, 2005;Sahin et al, 2008). Relocalization at the ER/Golgi compartment from the PM was also observed when the second signal of several known MYRed proteins was abolished, as in the Fyn and Yes protein kinases, in which the Cys residues necessary for the PAL were substituted by a Ser or the polybasic domain of Src protein kinase was made shorter (McCabe and Berthiaume, 1999).…”

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

“…GRASP65 was first described by Barr, Warren and colleagues as a Golgi cisternal stacking factor using an in vitro Golgi reassembly assay. 49 However, RNAi studies have later shown that its main function may not be in cisternal stacking, [50][51][52] but in the formation and/or maintenance of the tubules connecting the stacks within the Golgi ribbon. 52 GRASP65 is a major mitotically-phosphorylated Golgi protein.…”

“…Previous work has shown that RNAi depletion of the single Drosophila GRASP homologue (dGRASP) or its partner dGM130 does not lead to an apparent separation of the paired Golgi stacks. 12,51 Furthermore, the single CtBP/BARS homologue that exists in Drosophila (dCtPB) has been shown to mediate transcription repression regulating embryonic development, [67][68] but thus far, no link to the Golgi organization has been reported.…”

Membrane adhesion dictates Golgi stacking and cisternal morphology

“…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). In Schizosaccharomyces pombe, depletion of yeast GRASP homolog Grh1 has no effect on Golgi stacking (22).…”