Abstract
The endoplasmic reticulum (ER) membrane protein Rft1 is a scramblase for the anionic glycolipid Man5GlcNAc2-PP-dolichol (M5-DLO), a key intermediate in the pathway of protein N-glycosylation. As a member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter superfamily Rft1 resembles the bacterial MurJ Lipid II flippase which has an analogous substrate, but the mechanism by which it translocates M5-DLO is not known. Here we used AlphaFold3 and Chai1 to develop conformational models of yeast Rft1-M5-DLO complexes. The models suggest an alternating access transport mechanism with inward-open, occluded, and outward-facing states. A central cavity accommodates the hydrophilic M5-DLO head group, while the dolichol lipid tail is guided through a portal formed between transmembrane helices 1 and 8 to a hydrophobic groove outside the cavity. Comparative mutational analysis of cavity residues revealed a significant mechanistic divergence from MurJ which strictly relies on a conserved charged triad to anchor Lipid II via its pyrophosphate neck. Whereas mutations to these residues in MurJ result in total loss of function, Rft1 showed high tolerance to most charge inversion mutations in the central cavity when tested for function using a yeast reporter strain. This tolerance implies greater flexibility in Rft1's electrostatic requirements for engaging M5-DLO. Our findings suggest that Rft1 functions as a specialized, alternating access transporter for M5-DLO and identify key molecular determinants that are essential for function. The alternating access mechanism distinguishes Rft1 from all currently known scramblases which make use of a hydrophilic transmembrane groove to provide a pathway for lipid transit.