Mechanism of GRASP65 Mediated Organelle Tethering and its Regulation
2010-11-01T00:00:00Z (GMT) by
In higher eukaryotes, the Golgi apparatus is organized into a single copy, ribbon-like membrane network. The ribbon-like structure is established by lateral homotypic interactions between analogous cisternae in adjacent ministacks. Lateral linking may involve homotypic tethering followed by membrane fusion. Indeed, ribbon formation is blocked by depletion of the membrane tethering proteins GRASP65 and GRASP55, which are localized to cis and medial Golgi cisternae, respectively. In this thesis we present a structure-function analysis of GRASP65 in order to further understand its mechanism of action and regulation in Golgi ribbon formation. Because GRASP65 homo-oligomerizes in vitro we hypothesized that its self-interaction links cis cisternae prior to fusion. To test this model and determine the mechanism of GRASP65 selfinteraction we developed a cell-based organelle-tethering assay. GRASP65 was targeted to the mitochondrial outer membrane allowing a quantitative visual assessment of induced mitochondrial tethering. We observed that GRASP65 interacts in trans to tether organellar membranes, and the tethering involves the binding groove of the first of two PDZ-like domains present at its N-terminus. Tethering also required membrane anchoring of the PDZ domain suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. These results identify a homotypic PDZ interaction mediating organelle tethering in living cells. GRASP65 self-interaction is regulated by mitotic phosphorylation but the mechanism is unclear. In fact, the known GRASP65 phosphorylation sites are outside the self-interacting N-terminal domain, and their mutation to mimic phosphorylation failed to block tethering. We identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phospho-inhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.