Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole

Water expulsion by the contractile vacuole in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a-effector, Rab8a, the exocyst complex and LvsA, a protein of the Chediak-Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder suggesting that a Rab8a GEF activity is associated with the complex. Absence of Drainin leads to over-tethering and blocks fusion, while expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in post-fusion de-tethering. Interestingly, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype