Abstract
Monogalactosyl diacylglycerol (MGDG) is a major membrane lipid component in plants and is crucial for proper thylakoid functioning. However, MGDG in mammals has not received much attention, partly because of its relative scarcity in mammalian tissues. In addition, the biosynthetic pathway of MGDG in mammals has not been thoroughly analysed, although some reports have suggested that UGT8, a ceramide galactosyltransferase, has the potential to catalyse MGDG biosynthesis. Here, we successfully captured the endogenous levels of MGDG in HeLa cells using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)-based lipidomics. Cellular MGDG was completely depleted in CRISPR/Cas9-mediated UGT8 knockout (KO) HeLa cells. Transient overexpression of UGT8 enhanced MGDG production in HeLa cells, and the corresponding cell lysates displayed MGDG biosynthetic activity in vitro. Site-directed mutagenesis revealed that His358 within the UGT signature sequence was important for its activity. UGT8 was localized in the endoplasmic reticulum and activation of the unfolded protein response by membrane lipid saturation was impaired in UGT8 KO cells. These results demonstrate that UGT8 is an MGDG synthase in mammals and that UGT8 regulates membrane lipid saturation signals in cells.