PerspectiveAll the scramblase candidate deletion strains that we tested gave similar results in the CPY glycosylation screen. Whereas these proteins appear not to be necessary for GPD scrambling in vivo, it remains a possibility that their activity in vivo is obscured by redundancy as noted for ER glycerophospholipid scramblases where several proteins have been recently identified in mammalian cells. This could be tested by analyzing CPY glycosylation in strains lacking several of these proteins simultaneously, or by purifying the proteins and testing whether they are sufficient to promote GPD scrambling after reconstitution into vesicles. A similar test could be done to test whether the GPD synthase (Alg5) or the glucosyltransferases (Alg6, Alg8) are themselves GPD scramblases - the loss of the enzymatic activity of these proteins in the corresponding deletion strains would have obscured their possible scramblase activity in CPY blots. Finally, several of our scramblase candidates are essential proteins - these remain to be tested using temperature-sensitive strains.Molecular mechanism of glucosylation of the dolichol-linked N-glycan precursorAnant K. MenonReferences1) Schenk, B., Fernandez, F., and Waechter, C.J. (2001). The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum. Glycobiology 11, 61R-70R.2) Turco, S.J., Stetson, B., and Robbins, P.W. (1977). Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro. Proc Natl Acad Sci USA 74, 4411-4414.3) Rush, J.S., van Leyen, K., Ouerfelli, O., Wolucka, B., and Waechter, C.J. (1998). Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain. Glycobiology 8, 1195-1205.4) Pellegrini, M., Marcotte, E.M., Thompson, M.J., Eisenberg, D., and Yeates, T.O. (1999). Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci USA 96, 4285-4288.5) Dey, G., Jaimovich, A., Collins, S.R., Seki, A., and Meyer, T. (2015). Systematic Discovery of Human Gene Function and Principles of Modular Organization through Phylogenetic Profiling. Cell Rep 10, 993-1006.6) Skrabanek, L.A., and Menon, A.K. (2018). Phylogenetic profiling identifies glucosyl-phosphoryl dolichol scramblase candidates. bioRxiv, doi.org/10.1101/209106.86
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