Rapid degradation of GRASP55 and GRASP65 reveals their immediate impact on the Golgi structure

Abstract

GRASP65 and GRASP55 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, loss of gene function approaches by RNAi or gene knockout to dissect their respective roles often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for the induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the Golgi ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115 and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, neither GRASP55 and/or GRASP65 are required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking, but are involved in maintaining the integrity of Golgi ribbon together with GM130 and Golgin-45.