Proposed mechanism for Rft1-mediated scrambling of a dolichol-linked oligosaccharide

Abstract The membrane protein Rft1 is proposed to play an essential role in yeast and human cells by scrambling the glycolipid Man5GlcNAc2-PP-dolichol (M5-DLO) across the endoplasmic reticulum (ER) for protein N-glycosylation. While this activity has been demonstrated in liposomes reconstituted with purified Rft1, biochemical evidence of additional M5-DLO scramblases and the viability of Rft1-null Trypanosoma brucei suggest that scrambling may be a moonlighting function of Rft1 rather than its essential cellular role. To investigate this paradox, we used AlphaFold3 and Chai-1 to model the conformational dynamics of yeast Rft1-M5-DLO complexes. The models suggest an alternating access mechanism, typical of Multidrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) superfamily transporters, in which a cationic central cavity coordinates the anionic headgroup of M5-DLO, while the dolichol tail of the lipid is accommodated through a lateral portal formed by two transmembrane helices. We used the models to design mutations to disrupt the interaction between Rft1 and the M5-DLO headgroup, and to engineer a salt bridge to block the portal and stall transport. Using a Tet-off yeast reporter strain, we tested 26 central cavity mutants and identified two that supported cell growth poorly despite being well-expressed. Strikingly, the portal-blocking mutant which lacks scramblase activity supported robust growth. These data suggest that while M5-DLO binding is important for Rft1 essential function, scrambling activity is dispensable. We propose that Rft1 essential role may be as an M5-DLO chaperone, capturing and routing M5-DLO propitiously on the cytoplasmic side of the ER to coordinate DLO biosynthesis. Competing Interest Statement The authors have declared no competing interest. Footnotes Significant differences between this version and previous versions of the manuscript: Title updated: The title has been changed from "Proposed mechanism for Rft1-mediated scrambling of a dolichol-linked oligosaccharide" to "Mechanism-guided mutagenesis of Rft1 to test its role as a dolichol-linked oligosaccharide scramblase in cells" . Authors updated: Hannah G. Wolfe has been added to the author list . Text revisions: The Abstract, Introduction, Results, and Discussion sections have been substantively rewritten to frame the study as a test of competing hypotheses regarding Rft1's essential cellular function . Additionally, the alternating-access mechanism is now explicitly discussed as a "proposed" or "hypothetical" pathway . New evolutionary analysis added: A new structure-guided maximum likelihood phylogeny of the MOP transporter superfamily has been added as Figure S1, which explicitly positions Rft1 relative to MurJ and other MOP transporters . New quantitative and functional data added: New quantitative data assessing the physiological abundance of Rft1 in cells has been added to an updated Figure 4 . To unmask the physiological impact of targeted substitutions and account for "functional reserve," new high-resolution growth and glycosylation analyses using promoters of varying strengths (PGPD, PADH, and PRFT1) were added as new Figures S7, S8, and S9 . Figures and Supplemental files updated: Main Figures 4, 5, 6, and 8 have been revised . The supplementary files have been updated with the addition of new Figures S1, S5, S6, S8, and S9, and the update of the previous Figure S4 into new Figure S7