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

Abstract

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>de novo</jats:italic> 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.</jats:p>

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

“…This is directly reflected in the degree of sequence variability observed inside the binding pocket of PDZ1 and in the overall degree of frustration on the cleft between PDZ1 and PDZ2 (Figure 2). Since the coordinated action of these four proteins is necessary for the Golgi ribbon formation [ 22,23 ], this might have been the birth of the Golgi ribbon (Figure 8B).…”

“…In scenario 1, when native conditions were present, GRASPs would work as tethering factors capable of mediating several different PPIs essential for the correct flow of the exocytic pathway. They would also participate in the formation of the higher-order architecture of the Golgi in synergy with the Golgins [ 14,22,23 ]. The latter property might have been the natural cause of GRASP duplication since GRASP65 is responsible for retaining GM130 and p115 into the cis-Golgi.…”

“…The latter property might have been the natural cause of GRASP duplication since GRASP65 is responsible for retaining GM130 and p115 into the cis-Golgi. At the same time, GRASP55 in the trans/medial Golgi faces is responsible for the retention of Golgin45 [ 22 ]. The need for two GRASPs in Metazoans is also justified by the unique participation of these proteins in the Golgi ribbon formation (a vertebrate-specific structural organization [ 76 ]) and as a negative regulator of exocytic transport, necessary for correct protein-cargo glycosylation [ 77 ].…”

“…GRASP55 and 65 are necessary to anchor GM130 and Golgin45 to the Golgi. Moreover, these four proteins are also essential for the formation and integrity of the Golgi ribbon [ 22,23 ]. Although membrane anchoring has proved essential for GRASP tethering, the general occurrence of myristoylation in eukaryotes besides mammalians remains obscure.…”

Structure modeling hints at a granular organization of the Vertebrate Golgi ribbon

“…First, understanding of the contributions of individual components of the complex molecular machinery involved in ribbon formation and maintenance is incomplete (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Second, a lack of cellular systems where the detailed structure of the ribbon is intimately linked to a quantifiable biological function has prevented much experimental probing of the effects of this Golgi organization on a measurable biological output.…”