The role of dynamin-related proteins in vacuole biogenesis in fission yeast (Schizosaccharomyces pombe) : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

Dynamins are GTPases concerned with membrane tubulation and scission (Praefcke and McMahon, 2004). In the fission yeast, Schizosaccharomyces pombe, the dynamin-related proteins (DRPs) Vps1 and Dnm1 act redundantly in peroxisome biogenesis (Jourdain et al., 2008) but nothing is known about their other cellular roles. Fission yeast cells contain ~20 small, spherical vacuoles that undergo fission or fusion in response to environmental signals (Bone et al., 1998). S. pombe cells lacking Vps1 had smaller vacuoles with reduced capacity for fusion in response to hypotonic stress but enhanced fission in response to hypertonic conditions. Unlike wild type, vps1Δ vacuoles showed no change in diameter in response to temperature stress. Vps1-Cgfp localised to the vacuolar membrane both in living cells and in isolated vacuoles. vps1Δ cells showed close to wild type levels of vacuole protein processing and normal actin organisation and endocytosis. Overexpression of Vps1 caused a global transformation of vacuoles from spherical to tubular. Spherical vacuoles were restored by repression of vps1 expression or by induction of vacuole fusion. Tubulation was blocked in the presence of GTPγS and in a vps1 mutant that lacked the entire GTPase domain. Vacuole tubulation was more extensive in the absence of a second DRP, Dnm1. The absence of Dnm1 abolished the hyper fission phenotype of vps1Δ, whereas overexpression of Dnm1 induced vacuole fission. These results are consistent with a model of vacuole fission in which Vps1 creates a tubule of an appropriate diameter for subsequent scission by another DRP. Preliminary evidence suggests that Dnm1 serves the latter role.