We performed advanced lipidomics analyses of Sac1 degraded human A431 cells, including whole cell lipidomics, phosphoinositide-mass spectrometry and CLICK-lipid analysis, at different time points upon depletion of Sac1. This showed rapid changes of lipid levels, including PI4P, cholesterol and cholesteryl esters, at early timepoints after Sac1 depletion. Additionally, we performed advanced microscopy analysis using lipid biosensors and antibodies to assess the subcellular distribution of these lipid changes. The main findings show a rapid increase in PI4P in the trans-Golgi network (TGN), followed by a decrease in TGN cholesterol levels within 2 hours after Sac1 degradation.
Next, we investigated the effects of Sac1 depletion on Golgi integrity, showing rapid fragmentation of the Golgi stacks within 2 hours after Sac1 depletion by electron microscopy and immunofluorescence analysis. Whole cell proteomics of Sac1 degron A431 cell lysates showed major changes in glycosylation enzymes and Golgi structural proteins. Here we found that selective degradation of a subset of TGN proteins (including TGN46 and B4GALT1) occurs rapidly upon Sac1 depletion. Over time, this leads to terminal glycosylation defects. Interestingly, rapidly after Sac1 depletion we observed deacidification of the TGN, caused by disassembly of Golgi V-ATPase complex. Using molecular dynamic simulations, we showed that the changing PI4P and cholesterol distribution in the TGN, caused by Sac1 depletion, promotes a conformational change in the V0a2 subunit of the Golgi V-ATPase complex. This conformation inhibits V0 and V1 complex assembly and hence V-ATPase activity.
Additionally, we established an auxin-inducible degradation model targeting Sac1 in human Embryonic Stem Cells (hESCs). We differentiated the hESCs into different lineages, including neuronal cells, embryoid bodies and trophoblasts, from which we could successfully degrade Sac1. As Sac1 KO in mouse is embryonically lethal and loss-of-function of the SACM1L gene is not well tolerated in humans, we differentiated Sac1 degron cells into trophoblasts, which secrete human chorionic gonadotropic (hCG), important for implantation and gestation. We were able to recapitulate the main phenotypes of Sac1 degron A431 cells in trophoblasts. Additionally, Sac1 degradation in trophoblasts caused processing and secretion defects of hCG, implicating a physiological relevant role of Sac1 in early embryonic development.
