Constitutive signaling by an endosomal Rab5 isoform causes lipid metabolism dysfunction and ‘obesity’ in S. cerevisiae

Yeast cells build lipid droplets (LDs) in anticipation of starvation. LDs consist of amphipathic lipid monolayers surrounding a neutral lipid interior. Surface proteins allow lipid droplets to dock with other cellular compartments, but the unusual structure of LDs prevents compartments from fusing together. Instead, LDs are thought to dock with other organelles in order to enable lipid exchange between organelles. Identifying the proteins that mediate interactions between LDs and other organelles is a priority for understanding lipid metabolism and transport. The Rab GTPase Vps21 (yeast ortholog of human Rab5) controls endosomal tethering and vesicle fusion. In the signaling-active, GTP-bound state, Vps21 interacts with a set of effector proteins that function as endosome membrane tethers. The Vps21 mutant Vps21Q66L cannot hydrolyze GTP and is therefore ‘locked’ in the active, GTP-bound state and (it is thought) locked in interactions with effector protein tethers. We analyzed yeast cells expressing signaling-active Vps21Q66L using quantifiable enzyme-coupled endosomal cargo transport assays (e.g. CPY-invertase and Sna3- FLuciferase) and were surprised to find little to no misregulation of endosomal cargo transport. However, we observed vps21Q66L mutants display a pronounced cellular growth defect compared to wild type VPS21 cells. Because the vps21Q66L growth defect first as yeast exit logarithmic phase and should begin adaptation to starvation conditions, we investigated whether or not vps21Q66L mutants correctly build LDs. Using the LD marker Erg6-GFP, we observed by fluorescence microscopy that vps21Q66L mutants display higher LD signal than wild type cells, and vps21Q66L LDs possess an aberrant, distended LD morphology. Biochemical analysis (gas chromatography-mass spectrometry) of lipid content from vps21Q66L mutants indicates 55 5th Annual Student Research Symposium Meeting of the Minds Event Program an ‘obese’ phenotype in which cells accumulate an overabundance of neutral lipids. These phenotypic data suggest a new link between Rab signaling at endosomes and cellular lipid metabolism. Ongoing studies are investigating whether the obese phenotype is a result of increased synthesis of neutral lipids or slower consumption. Also, we are pursuing both genetic and biochemical approaches to identify proteins that interact with GTP-bound Vps21 to trigger aberrant lipid metabolism and LD morpholog