Topological Analysis of Niemann-Pick C1 Protein Reveals That the Membrane Orientation of the Putative Sterol-sensing Domain Is Identical to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol Regulatory Element Binding Protein Cleavage-activating Protein

doi:10.1074/jbc.M002184200

Topological Analysis of Niemann-Pick C1 Protein Reveals That the Membrane Orientation of the Putative Sterol-sensing Domain Is Identical to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol Regulatory Element Binding Protein Cleavage-activating Protein*

From the Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029

Abstract

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.

Footnotes

↵* These studies were supported in part by a grant from the Ara Parseghian Medical Research Foundation and a grant from the March of Dimes Foundation.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.
↵‡ To whom correspondence should be addressed: Dept. of Human Genetics, P. O. Box 1498, Mount Sinai School of Medicine, Fifth Ave. at 100th St., New York, NY 10029. Tel.: 212-659-6720; Fax: 212-348-3605; E-mail: Ioanny01@doc.mssm.edu.
Published, JBC Papers in Press, May 19, 2000, DOI 10.1074/jbc.M002184200
Abbreviations:

NPC

Niemann-Pick type C

HMG-R

3-hydroxy-3-methylglutaryl-coenzyme A reductase

SCAP

sterol regulatory element binding protein cleavage-activating protein

ER

endoplasmic reticulum

SSD

sterol-sensing domain

TGN

trans-Golgi network

PTC

Patched

TM

transmembrane

wt

wild-type

PAGE

polyacrylamide gel electrophoresis

α-Gal A

α-galactosidase A

FITC

fluorescein isothiocyanate

PBS

phosphate-buffered saline

BSA

bovine serum albumin
- Received March 15, 2000.
- Revision received April 26, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.