The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

doi:10.1074/jbc.274.14.9627

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo*

From the ^tOaLipid Cell Biology Section, Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the ^tObDivision of Neonatology, Department of Pediatrics, Georgetown University Medical Center, Washington, D. C. 20007, the ^tOcNeurobiology Research Laboratory, Veterans Affairs Medical Center, Newington, Connecticut 06111, the ^tOdDevelopmental and Metabolic Neurology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892, ^tOeTottori University, Faculty of Medicine, Yanago, Japan 683-8503, the ^tOfSaccomanno Research Institute, Saint Mary’s Hospital, Grand Junction, Colorado 81502, the ^tOgDepartment of Pediatrics and Biological Structure, University of Washington, Seattle, Washington 98195, the ^tOhDepartment of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the ⁱDepartment of Biochemistry and INSERM U 189, Faculte de Medecine, Lyon-Sud, Oullins, France 69921, and the ^tOjDepartment of Neurology, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Abstract

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking.

Immunocytochemical studies in fibroblasts reveal that theNPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(−) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 °C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation.

Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [¹⁴C]sucrose. Compartmental modeling of the observed [¹⁴C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [¹⁴C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein.

We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Footnotes

↵* 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.
↵FNk To whom correspondence should be addressed: Lipid Cell Biol. Section, Bldg. 8, Rm. 427, NIDDK, National Institutes of Health, 8 Center Dr., MSC 0850, Bethesda, MD 20892. Tel.: 301-496-2050; Fax: 301-402-0723; E-mail: joanbm{at}bdg8.niddk.nih.gov.
↵2 E. B. Neufeld, M. Wastney, S. Patel, S. Suresh, A. M. Cooney, N. K. Dwyer, C. F. Roff, K. Ohno, J. A. Morris, E. D. Carstea, J. P. Incardona, J. F. Strauss III, M. T. Vanier, M. C. Patterson, R. O. Brady, P. G. Pentchev, and E. J. Blanchette-Mackie, unpublished data.
↵3 S. Patel, manuscript in preparation.
↵4 E. J. Blanchette-Mackie, manuscript in preparation.
↵5 J. Incardona, unpublished observations.
Abbreviations:

NP-C

Niemann-Pick C disease

G_M2

GalNAcβ1–4[NeuAcα2–3]Galβ1–4Glcβ1-Cer

LAMP

lysosome-associated membrane protein

LDL

low density lipoprotein

LPDS

lipoprotein-deficient serum

MPR

mannose 6-phosphate receptor

NPC1

Niemann-Pick C1 protein

LRSC

lissamine rhodamine sulfonyl chloride
- Received November 4, 1998.
- Revision received January 5, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.