Background: Excess oleate induces triacylglycerol synthesis and proliferation of lipid particles (LP) in yeast. Results: Oleate-induced LP formation is dependent on the glycerol-3-phosphate acyltransferase (GPAT) isoform Gat1p, which physically interacts with LPs. Conclusion: Formation of oleate-induced LPs depends on active Gat1p (but not Gat2p). Significance: Fatty acid surplus is controlled at the GPAT step with unique contributions from different isoforms.