Periodic Reporting for period 1 - PIP-AID (Characterization of the role of Sac1 in the spatiotemporally controlled interplay between PI4P and cholesterol homeostasis)
Periodo di rendicontazione: 2022-11-01 al 2025-07-31
Phosphatidylinositol 4 phosphate (PI4P) is the most abundant phosphoinositide lipid species in the cell. It is asymmetrically distributed over the cell and has essential functions in secretory trafficking, membrane identity and lipid metabolism. Disruptions in cellular PI4P levels can give rise to neuro-degenerative diseases including ALS and Alzheimer’s disease. PI4P levels are regulated by an interplay between PI4-kinases and the integral ER/Golgi phosphatase Sac1. Sac1 tightly controls distinct PI4P membrane pools, maintaining the asymmetrical distribution of PI4P through the cell. This is important to fuel non-vesicular lipid transfer at membrane contact sites, mediated by lipid transfer proteins. e.g. OSBP, which mediates the transport of cholesterol from the ER to the Golgi, in exchange for PI4P. Sac1 is the only known PI4P phosphatase and is essential for cell viability. Sac1 knockout in mice or fruit fly results in early embryonic lethality. In this study we used auxin-inducible degradation of Sac1, to assess the acute effects of Sac1 loss.
Overall, we aimed to contribute to a deeper and systematic understanding into the regulatory role of Sac1 in lipid metabolism and Golgi functioning and provide a new means to investigate the unexplored role of Sac1 in development.
Overall, we aimed to contribute to a deeper and systematic understanding into the regulatory role of Sac1 in lipid metabolism and Golgi functioning and provide a new means to investigate the unexplored role of Sac1 in development.
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.
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.
Overall, the project resulted in two scientific publications. The results reveal that the assembly of the Golgi V-ATPase is regulated by the TGN membrane lipid composition via Sac1 fuelled cholesterol/PI4P exchange. During the project, the auxin-inducible degradation of Sac1 has been implemented in hESCs, which could be differentiated into different cell lineages. Sac1 degradation in human differentiated trophoblasts showed processing defects of chorionic gonadotropin, which is in line with loss-of-function intolerance of the human SACM1L gene. The auxin-inducible degradation system can be applied to other target proteins, opening new avenues of research into essential proteins in specialized cells.
How the lipid interactions of the V-ATPase complex affect its activity and how Sac1 mediates the selective degradation of TGN proteins are important topics for future studies.
How the lipid interactions of the V-ATPase complex affect its activity and how Sac1 mediates the selective degradation of TGN proteins are important topics for future studies.