dGRASP localization and function in the early exocytic pathway in Drosophila S2 cells.
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.
The Cell Microscopy Centre, Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, AZU, 3584CX Utrecht, The Netherlands.
Vangelis
V
Kondylis
Kirsten M
KM
Spoorendonk
Catherine
C
Rabouille
D000818:Animals; D001324:Autoantigens; D002460:Cell Line; D029721:Drosophila Proteins / Q000378:metabolism* / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology
D001324:Autoantigens; D029721:Drosophila Proteins; C506558:GRASP65 protein, Drosophila; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins