Conformational changes in the Niemann-Pick Type C1 protein NCR1 drive sterol translocation

The membrane protein Niemann-Pick Type C1 protein (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking and the mechanism for sterol translocation has remained unclear. Here we provide evidence that a glycocalyx is indeed present inside isolated Saccharomyces cerevisiae vacuoles, and report four cryo-EM structures of NCR1 in two distinct conformations that elucidate how it moves sterol through the glycocalyx. The two conformations, named "tense" and "relaxed", illustrate movement of sterol through a tunnel formed by the luminal domains. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the transmembrane domains form a motor-unit that sequentially adopts a tense and relaxed conformation to drive changes in luminal/extracellular domains. SIGNIFICANCE STATEMENTNiemann-Pick Type C1 (NPC1, named NCR1 in yeast) proteins play a critical role in sterol homeostasis by facilitating the integration of sterols into membranes of acidic organelles like lysosomes and vacuoles. The inner surface of these organelles membranes is shielded by the glycocalyx. Here, we provide evidence that a glycocalyx is present in vacuoles from Saccharomyces cerevisiae and demonstrate that NCR1 transports sterols across it by undergoing conformational changes. Our structures suggest a transport model where sterol transport is linked to proton-driven changes in the transmembrane region. This work sheds light on the mechanism of NPC1 protein function and has broad implications for understanding lysosomal storage disorders and for mechanisms employed by members of the Resistance-Nodulation-Division (RND) superfamily.