2025/12/09 by George N. Chiduza, Kentaro Sakata, Hannah G. Wolfe +2 · 1 voice
Biochemistry, Genetics and Molecular Biology · #Bacterial Genetics and Biotechnology #Cellular transport and secretion #Fungal and yeast genetics research
paper · doi:10.64898/2025.12.07.692794
openalex publication_date 2025/12/09 · openalex created_date 2025/12/10 · openalex updated_date 2026/07/14
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’s essential function, scrambling activity is dispensable. We propose 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. Importance Cell surface and secreted proteins are decorated with sugar chains. These chains are first assembled on a lipid carrier. Initial stages of assembly occur on the cytoplasmic side of a subcellular structure called the endoplasmic reticulum (ER). To complete assembly, the partially assembled lipid-linked sugar chain must be flipped across the ER. Here we use computationally guided cell-based assays to examine the role of the Rft1 protein in this process.