Abstract
Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(deltaLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.
Highlights
Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder that is characterized by endosomal accumulation of low-density lipoprotein (LDL)-derived cholesterol (Patterson et al, 2001) and is caused by mutations in NPC1 or NPC2 (Carstea et al, 1997; Naureckiene et al, 2000)
Cholesterol depletion facilitated ubiquitylation of NPC1 expressed in COS cells NPC1 ubiquitylation was examined in COS cells transfected with Flag-NPC1 and myc/His6-ubiquitin. 48 hours after transfection, cells were incubated either in cholesterol-rich medium [Dulbecco’s modified Eagle’s medium (DMEM) + 10% bovine calf serum (BCS)] or cholesterol-depleted medium [DMEM + 10% lipoproteindeficient serum (LPDS)] supplemented with compactin and mevalonate
When cells were incubated in the cholesteroldepleted medium, the total cellular cholesterol level decreased in a time-dependent manner to reach ~60% of the control level at 12 hours
Summary
Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder that is characterized by endosomal accumulation of low-density lipoprotein (LDL)-derived cholesterol (Patterson et al, 2001) and is caused by mutations in NPC1 or NPC2 (Carstea et al, 1997; Naureckiene et al, 2000). NPC1 is a membrane protein that resides primarily in the late endosome (Higgins et al, 1999), whereas NPC2 is a soluble protein that resides primarily in the lysosome (Vanier and Millat, 2004) These two proteins have been shown to function in the same endosomal cholesterol efflux pathway (Sleat et al, 2004). Solution of the human NPC1 membrane topology revealed a 1278 amino acid protein with 13 transmembrane domains and a sterol-sensing domain (SSD) located from transmembrane domains III to VII (Davies and Ioannou, 2000) This protein has been shown to possess a lipid permease activity (Davies et al, 2000) and the functional significance of its SSD has been well documented (Watari et al, 1999a; Millard et al, 2005), it is not known whether NPC1 function is regulated by cellular cholesterol. One might hypothesize a cholesteroldependent regulation of NPC1 function, either by its association with other proteins or by post-translational modifications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
