Sterol transport by the NPC system in yeast
Cholesterol is an essential element of cellular membranes and is a precursor for a variety of sterols, some of which act as hormones. Cholesterol is synthesized in the smooth endoplasmic reticulum, but is also obtained from dietary sources and recovered from lipid droplets. which serve as a storage compartment. The lysosome plays a pivotal role in trafficking of sterols with the NPC system ultimately transporting these amphiphilic molecules through the glycocalyx and depositing them in the membrane. The NPC system consists of two components and is named after Niemann-Pick disease resulting from cholesterol deficiency. NPC2 is a soluble carrier protein that binds cholesterol within the lysosome and transfers it to NPC1, which moves it into the membrane. In collaboration with the laboratory of Bjørn Pedersen at Aarhus University, we study the yeast homolog of NPC1, NCR1. In yeast, the primary sterol is ergosterol and the vacuole plays a role analogous to the lysosome.
NCR1 has two copies of the RND fold
The Resistance-Nodulation-Division (RND) family includes a variety of membrane transporters, the best known of which is the multidrug resistance protein AcrB. The family is characterized by the RND fold consisting of 5+1 transmembrane helices separated by an extracellular “sandwich” domain. The proteins typically oligomerize with AcrB forming a homotrimer. NCR1 and its human homolog NPC1 consist of two tandem copies of the RND fold, thus displaying pseudo-symmetry within a single polypeptide. In addition, an N-terminal domain (NTD) is present in the lumen and tethered to the membrane by an additional transmembrane helix (TM1). The NTD initially receives sterol the soluble component NPC2. Although the RND pseudo repeats are firmly associated with one another, the NTD and TM1 appear to be more loosely bound to the core of the protein.
NCR1 structure reveals a hydrophobic tunnel for sterol transport
The Pedersen lab has solved structures of NCR1 and NPC2, the former revealing the juxtaposition of the RND domains and the NTD and the latter shedding light on the transfer process. The structure of NCR1 also reveals a continuous tunnel that runs from the sterol binding site in the NTD, between the pseudo-symmetric lumenal domains (MLD and CTD) and into the lumenal leaflet of the bilayer. This tunnel - shown in the inset on the lower left - is postulated to provide a pathway for carrying sterol through the glycocalyx which is presumed to line the lumenal surface of the vacuole.
Mechanistic model for sterol transport
Current structures from the Pedersen laboratory and others give rise to a model for sterol transport by the NPC system. The cycle starts by loading NPC2 with sterol within the lumen of the vacuole or lysosome. NPC2 then transfers the sterol to the NTD of NCR1 (or NPC1 in lysosomes). After dissociation of this complex, the NTD transfers the sterol into the tunnel between MLD and CTD domains, where it passes across the glycocalyx to reach the lumenal leaflet of the membrane. The sterol is then released from the so-called sterol sensing domain composed by the first set of transmembrane helices (TM2-7).
Acidic pair may hold the key for energy coupling
A conserved pair of acidic residues is seen at the interface between transmembrane domains of many RND proteins (D631 & E1068 for Ncr1). This pair is generally flanked by a basic residue, in this case His1072. Although proton coupling is well established for multidrug resistance RND transporters, the driving force for sterol transport is not yet established. Recent cryo-EM structures of NCR1 at different pHs shows that the H-bonding of this acidic pair is switched from the His to a Lys and that associated allosteric movements of the RND domains are associated with movement of sterol through the tunnel. This led to a proposed mechanism by which pH driven conformational change gives rise to sterol transport across the glycocalyx.
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Winkler MBL, Kidmose RT, Szomek M, Thaysen K, Rawson S, Muench SP, Wüstner D, Pedersen BP. Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins. Cell. 2019 Oct 3;179(2):485-497.e18. doi: 10.1016/j.cell.2019.08.038.
Zhang XC, Liu M, Han L. Energy coupling mechanisms of AcrB-like RND transporters. Biophys Rep. 2017;3(4):73-84. doi: 10.1007/s41048-017-0042-y.
Frain KM, Dedic E, Nel L, Bohush A, Olesen E, Thaysen K, Wüstner D, Stokes DL, Pedersen BP. Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation. Proc Natl Acad Sci U S A. 2024 Apr 9;121(15):e2315575121. doi: 10.1073/pnas.2315575121