Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization

Abstract

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoproteinderived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal͞endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesteroltrafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the Cterminal 4-aa residues containing the LLNF lysosometargeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.

Targeting of NPC1 to Late Endosomes Involves Multiple Signals, Including One Residing within the Putative Sterol-sensing Domain

“…2), in agreement with a previous report (15) where deletion of the C-terminal 4 amino acids of the cytoplasmic tail ablated NPC1 location and function. Inspection of the sequence surrounding the dileucine motif revealed that the C-terminal 8 amino acids of the tail contains a di-acidic sequence (ERE) conforming to the DXE motif proposed to elicit efficient COPII-mediated ER export of certain transmembrane proteins (18, 26 -29).…”

“…It is composed of 13 transmembrane domains and a small cytoplasmic tail (14) that has been suggested to be essential for the transport of newly synthesized NPC1 to the late endosome (15). Examination of this cytoplasmic tail (numbered 1-27 from Gly 1252 ) reveals several potential late endosome targeting regions, such as a dileucine motif at positions 24 and 25, a tyrosine at position 16, a cysteine at position 10, and a putative ER maturation signal 20 ERE 22 (Fig.…”

“…NPC1 contains a putative dileucine motif at the C terminus of a small cytoplasmic tail (14). A 4-amino acid truncation of the cytoplasmic tail removing the dileucine motif abolishes NPC1 protein function; therefore, it was presumed that this motif is responsible for sorting NPC1 to the E/L pathway (15). A more recent study, however, has demonstrated that the NPC1 protein is capable of correcting the NPC1 phenotype even when devoid of the 27-amino acid cytoplasmic tail (14).…”

Localization of Niemann–Pick C1 protein in astrocytes: Implications for neuronal degeneration in Niemann– Pick type C disease

“…A trafficking pathway for delivery of nutrients and metabolites between astrocytes and neurons is supported by previous studies. Astrocytes have been implicated in the delivery of glucose and lactate to neurons (19) and are known to play an important role in the uptake and recycling of neuronally released glutamate (20). In this respect it is of interest that glutamine synthetase in human cerebral cortical astrocytes is localized in a manner that closely resembles the perisynaptic astrocytic localization of NPC1 in this study (16).…”

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

“…CLSM was performed 20 h after transfection using a 25 ϫ 0.8 NA oil immersion objective. Filipin staining was performed as described previously (Watari et al, 1999) on fixed cells expressing DiHcRED-MAL by using 5 g/ml filipin.…”