DIRC2 (Disrupted in renal carcinoma 2) has been initially identified as a breakpoint-spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the MFS (major facilitator superfamily) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine embryonic fibroblasts. The cleavage site within DIRC2 was mapped between amino acid residues 214 and 261 using internal epitope tags, and is presumably located within the tentative fifth intralysosomal loop, assuming the typical MFS topology. Lysosomal targeting of DIRC2 was demonstrated to be mediated by a N-terminal dileucine motif. By disrupting this motif, DIRC2 can be redirected to the plasma membrane. Finally, in a whole-cell electrophysiological assay based on heterologous expression of the targeting mutant at the plasma membrane of Xenopus oocytes, the application of a complex metabolic mixture evokes an outward current associated with the surface expression of full-length DIRC2. Taken together, these data strongly support the idea that DIRC2 is an electrogenic lysosomal metabolite transporter which is subjected to and presumably modulated by limited proteolytic processing.
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Research Article|
September 14 2011
Disrupted in renal carcinoma 2 (DIRC2), a novel transporter of the lysosomal membrane, is proteolytically processed by cathepsin L
Lalu Rudyat Telly Savalas;
*Biochemical Institute, Christian-Albrechts-University, Kiel, Germany
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Bruno Gasnier;
Bruno Gasnier
1
†Université Paris Descartes, Centre National de la Recherche Scientifique, UMR 8192 Paris, France
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Markus Damme;
Markus Damme
‡Department of Biochemistry, University of Bielefeld, Bielefeld, Germany
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Torben Lübke;
Torben Lübke
§Biochemistry II, University of Göttingen, Göttingen, Germany
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Christian Wrocklage;
Christian Wrocklage
∥Institute for Physiological Chemistry, Philipps University, Marburg, Germany
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Cécile Debacker;
Cécile Debacker
†Université Paris Descartes, Centre National de la Recherche Scientifique, UMR 8192 Paris, France
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Adrien Jézégou;
Adrien Jézégou
†Université Paris Descartes, Centre National de la Recherche Scientifique, UMR 8192 Paris, France
¶Graduate School ED 419, Université Paris-Sud 11, Kremlin-Bicêtre, France
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Thomas Reinheckel;
Thomas Reinheckel
**Institute for Molecular Medicine and Cell research and Centre for Biological Signalling Studies (bioss), Albert-Ludwigs-University, Freiburg, Germany
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Andrej Hasilik;
Andrej Hasilik
∥Institute for Physiological Chemistry, Philipps University, Marburg, Germany
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Paul Saftig;
Paul Saftig
3
*Biochemical Institute, Christian-Albrechts-University, Kiel, Germany
3Correspondence may be addressed to either of these authors (baschroeder@biochem.uni-kiel.de or psaftig@biochem.uni-kiel.de).
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Bernd Schröder
Bernd Schröder
3
*Biochemical Institute, Christian-Albrechts-University, Kiel, Germany
3Correspondence may be addressed to either of these authors (baschroeder@biochem.uni-kiel.de or psaftig@biochem.uni-kiel.de).
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Publisher: Portland Press Ltd
Received:
January 25 2011
Revision Received:
May 06 2011
Accepted:
June 22 2011
Accepted Manuscript online:
June 22 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 439 (1): 113–128.
Article history
Received:
January 25 2011
Revision Received:
May 06 2011
Accepted:
June 22 2011
Accepted Manuscript online:
June 22 2011
Citation
Lalu Rudyat Telly Savalas, Bruno Gasnier, Markus Damme, Torben Lübke, Christian Wrocklage, Cécile Debacker, Adrien Jézégou, Thomas Reinheckel, Andrej Hasilik, Paul Saftig, Bernd Schröder; Disrupted in renal carcinoma 2 (DIRC2), a novel transporter of the lysosomal membrane, is proteolytically processed by cathepsin L. Biochem J 1 October 2011; 439 (1): 113–128. doi: https://doi.org/10.1042/BJ20110166
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