Mechanism-guided mutagenesis of Rft1 to test its role as a dolichol-linked oligosaccharide scramblase in cells

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 problem, 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 2 that supported cell growth poorly despite being well-expressed. Strikingly, the portal-blocking mutant which is predicted to lack scramblase activity supported robust growth. These data suggest that while M5-DLO binding is important for Rft1's essential function, scrambling activity is dispensable. We speculate that Rft1's 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.