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J. Biol. Chem., Vol. 281, Issue 46, 35436-35445, November 17, 2006

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Demonstration of Lysosomal Localization for the Mammalian Ependymin-related Protein Using Classical Approaches Combined with a Novel Density Shift Method^*

Maria Cecilia Della Valle, David E. Sleat, Istvan Sohar, Ting Wen^¶, John E. Pintar^¶, Michel Jadot^||, and Peter Lobel¹

From the Center for Advanced Biotechnology and Medicine, Departments of Pharmacology and ^¶Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 and ^||Laboratoire de Chimie Physiologique, Unite de Recherche en Physiologie Moleculaire, Facultes Universitaires Notre-Dame de la Paix, 61 Rue de Bruxelles, 5000 Namur, Belgium

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.

Received for publication, June 29, 2006 , and in revised form, August 23, 2006.

^* This study was supported by National Institutes of Health Grant DK054317, a grant from the Ara Parseghian Medical Research Foundation (to P. L.), and Fonds de la Recherche Fondamentale Collective Grant 2.4505.01 (to M. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: CABM, 679 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-5032; Fax: 732-235-4466; E-mail: lobel{at}cabm.rutgers.edu.

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