c When deprived of nitrogen (N), the photosynthetic microalga Chlamydomonas reinhardtii accumulates large quantities of triacylglycerols (TAGs), making it a promising source of biofuel. Prominent transcriptional changes associated with the conditions leading to TAG accumulation have been found, suggesting that the key enzymes for TAG metabolism might be among those that fluctuate in their expression during TAG synthesis and breakdown. Using a Saccharomyces cerevisiae lipase null mutant strain for functional complementation, we identified the CrLIP1 gene from Chlamydomonas based on its ability to suppress the lipase deficiency-related phenotypes of the yeast mutant. In Chlamydomonas, an inverse correlation was found between the CrLIP1 transcript level and TAG abundance when Chlamydomonas cultures were reversibly deprived of N. The CrLIP1 protein expressed and purified from Escherichia coli exhibited lipolytic activity against diacylglycerol (DAG) and polar lipids. The lipase domain of CrLIP1 is most similar to two human DAG lipases, DAGL␣ and DAGL. The involvement of CrLIP1 in Chlamydomonas TAG hydrolysis was corroborated by reducing the abundance of the CrLIP1 transcript with an artificial micro-RNA, which resulted in an apparent delay in TAG lipolysis when N was resupplied. Together, these data suggest that CrLIP1 facilitates TAG turnover in Chlamydomonas primarily by degrading the DAG presumably generated from TAG hydrolysis. P lants, animals, and fungi accumulate triacylglycerols (TAGs) to store excess carbon and energy. For industrial biofuel production, triacylglycerol can be converted into fatty acid methyl esters (FAMEs), the major components of biodiesel, through transesterification (12). TAGs harvested from cells can thus be a source of biofuel. Thanks to their higher growth rate and higher oil content, microalgae can, in theory, produce TAG at yields surpassing those of plants (29). However, the details of lipid metabolism in algae are poorly understood.Chlamydomonas reinhardtii has a long history of serving as a model system for studying cell motility and photosynthesis (18,31,41). The recent development of genetic and molecular biological tools, such as nuclear genome transformation (21) and gene silencing with artificial micro-RNA (35), has also made Chlamydomonas an excellent model alga for a wider spectrum of research topics, including lipid metabolism and biofuel production. In Chlamydomonas, the glycerolipid composition was explored (16), and the lipid metabolism pathways have been annotated in silico (38). Certain enzymes for TAG metabolism have also been characterized experimentally (7). Like many other algae, Chlamydomonas accumulates TAG under nitrogen deprivation conditions (34, 52).In plants, TAG biosynthesis starts with the plastid production of fatty acids, which are then esterified to glycerol-3-phosphate, forming phosphatidic acid (PtdOH). The endoplasmic reticulum (ER) is another site for lipid assembly and fatty acid modification (44). Diacylglycerol is produced after the removal of...