Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.
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Research Article|
March 15 2006
Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes
Marielle Boonen;
Marielle Boonen
*URΦM, Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium
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Isabelle Hamer;
Isabelle Hamer
*URΦM, Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium
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Muriel Boussac;
Muriel Boussac
†Laboratoire de Chimie des Protéines, CEA/INSERM/UJF, F-38054 Grenoble, France
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Anne-Françoise Delsaute;
Anne-Françoise Delsaute
*URΦM, Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium
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Bruno Flamion;
Bruno Flamion
‡URΦM, Laboratoire de Physiologie/Pharmacologie, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium
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Jérôme Garin;
Jérôme Garin
†Laboratoire de Chimie des Protéines, CEA/INSERM/UJF, F-38054 Grenoble, France
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Michel Jadot
Michel Jadot
1
*URΦM, Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium
1To whom correspondence should be addressed (email michel.jadot@fundp.ac.be).
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Publisher: Portland Press Ltd
Received:
October 10 2005
Revision Received:
December 16 2005
Accepted:
December 20 2005
Accepted Manuscript online:
December 20 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (1): 39–47.
Article history
Received:
October 10 2005
Revision Received:
December 16 2005
Accepted:
December 20 2005
Accepted Manuscript online:
December 20 2005
Citation
Marielle Boonen, Isabelle Hamer, Muriel Boussac, Anne-Françoise Delsaute, Bruno Flamion, Jérôme Garin, Michel Jadot; Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes. Biochem J 1 April 2006; 395 (1): 39–47. doi: https://doi.org/10.1042/BJ20051647
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