TY - JOUR
T1 - Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation
AU - Frain, Kelly M.
AU - Dedic, Emil
AU - Nel, Lynette
AU - Bohush, Anastasiia
AU - Olesen, Esben
AU - Thaysen, Katja
AU - Wüstner, Daniel
AU - Stokes, David L.
AU - Pedersen, Bjørn Panyella
PY - 2024/4
Y1 - 2024/4
N2 - The membrane protein Niemann-Pick type C1 (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 supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. 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 proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.
AB - The membrane protein Niemann-Pick type C1 (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 supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. 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 proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.
KW - cryo-EM
KW - glycocalyx
KW - Niemann–Pick type C protein
KW - sterol uptake
KW - vacuole
KW - Sterols/metabolism
KW - Niemann-Pick C1 Protein/metabolism
KW - Membrane Glycoproteins/metabolism
KW - Saccharomyces cerevisiae/genetics
KW - Carrier Proteins/metabolism
KW - Natural Cytotoxicity Triggering Receptor 1/metabolism
U2 - 10.1073/pnas.2315575121
DO - 10.1073/pnas.2315575121
M3 - Journal article
C2 - 38568972
AN - SCOPUS:85190084482
SN - 0027-8424
VL - 121
SP - e2315575121
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 15
M1 - e2315575121
ER -