Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER- trans -Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
Abstract Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER- trans Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.
Abstract The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co‐migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement—including cell starvation—or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.
Abstract SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme’s catalytic activity as well as on its N -terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca 2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER.
