Triacylglycerols are important energy reservoirs in yeast. In addition, they serve important functions as precursors for membrane lipids and thus drive cellular growth and cell cycle progression. Cells lacking triacylglycerol synthesis are very sensitive against excess unsaturated fatty acids. On the other hand, increased lipolysis that results in excess diacylglycerols and free fatty acids, may induce lipotoxic effects. It was the aim of this study to identify factors that are responsible for fatty acid sensitivity.In a first experimental approach, Tgl4p, the yeast ortholog of mammalian adipose triglyceride lipase ATGL, was over-expressed in about 4700 single yeast deletion mutants. A total of 17 mutants were identified that were unable to grow upon Tgl4p over-expression. Among the affected genes were COA4, COX17, QCR9, YKT9 and LIP5, which play an important role in mitochondrial respiration. In addition, the transcription factors Ino2p/Ino4p, which play a central role in the regulation of phospholipid biosynthesis, were found to be essential to counteract over-expression of the Tgl4p lipase.A second approach was aimed at identifying genes that are responsible for oleate and palmitoleate-induced toxicity in a strain lacking triacylglycerol synthesis. In summary, these studies uncovered an unexpected link between lipolysis and mitochondrial function, and underscore the close regulatory interaction between phospholipid and neutral lipid synthesis, in yeast.