Role of Niemann-Pick Type C1 Protein in Intracellular Trafficking of Low Density Lipoprotein-derived Cholesterol

doi:10.1074/jbc.275.6.4013

Role of Niemann-Pick Type C1 Protein in Intracellular Trafficking of Low Density Lipoprotein-derived Cholesterol*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

Abstract

Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL36709 (to T. Y. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
↵‡ To whom correspondence should be addressed. Tel.: 603-650-1622; Fax: 603-650-1128; E-mail: Ta.Yuan.Chang@Dartmouth.edu.
↵2 In protocol B, after incubation for 4 to 5 h at 17–19 °C, 20–25% of [³H]CL-LDL was hydrolyzed in 25RA and CT cells. At 17–19 °C, with or without, a small percentage of [³H]CL-LDL (1–2% of total or 5–7% of hydrolyzed) could be reesterified to form [³H]cholesteryl oleate in 25RA and CT cells. This result is consistent with the observation (5, 30) that a small portion of cholesterol may be transported directly to ACAT for reesterification without traversing the PM. This result could also be explained by the finding that a small but significant percentage of ACAT may reside in a non-ER cytoplasmic organelle (31, 32).
Abbreviations:

NPC

Niemann Pick Type C

LDL

low density lipoprotein

PM

plasma membrane

CHO

Chinese hamster ovary

ER

endoplasmic reticulum

SCAP

SREBP cleavage-activating protein

ACAT

acyl-coenzyme A:cholesterol transferase

WT

wild-type

RT

reverse transcriptase

RACE

rapid amplification of cDNA ends

[³H]CL-LDL

[³H]cholesteryl linoleate-labeled LDL

[³H]CH/PC

[³H]cholesterol/phospholipid

GFP

green fluorescent protein

PBS

phosphate-buffered saline

bp

base pair

PCR

polymerase chain reaction

kb

kilobase

CD

cyclodextrin

hmNPC1

hamster NPC1
- Received October 28, 1999.
- Revision received November 16, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.