Until recently, a modest number of approx. 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. In the present study, we demonstrate the lysosomal localization of the mouse orthologue of the human C1orf85 protein, which has been termed kidney-predominant protein NCU-G1 (GenBank® accession number: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 (lysosome-associated membrane protein-1) as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells, the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild-type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown, by digesting the protein with N-glycosidase F, to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine residue for the tyrosine residue within the putative sorting motif.
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Research Article|
July 29 2009
NCU-G1 is a highly glycosylated integral membrane protein of the lysosome
Oliver Schieweck;
Oliver Schieweck
*Abteilung Biochemie II, Georg-August-Universität Göttingen, Göttingen 37073, Germany
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Markus Damme;
Markus Damme
*Abteilung Biochemie II, Georg-August-Universität Göttingen, Göttingen 37073, Germany
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Bernd Schröder;
Bernd Schröder
†Institut für Biochemie, Christian-Albrechts-Universität Kiel, Kiel 24118, Germany
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Andrej Hasilik;
Andrej Hasilik
‡Institut für Physiologische Chemie, Philipps-Universität Marburg, Marburg 35032, Germany
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Bernhard Schmidt;
Bernhard Schmidt
*Abteilung Biochemie II, Georg-August-Universität Göttingen, Göttingen 37073, Germany
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Torben Lübke
Torben Lübke
1
*Abteilung Biochemie II, Georg-August-Universität Göttingen, Göttingen 37073, Germany
1To whom correspondence should be addressed (email tluebke@gwdg.de).
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Publisher: Portland Press Ltd
Received:
April 08 2009
Revision Received:
May 07 2009
Accepted:
June 02 2009
Accepted Manuscript online:
June 02 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 422 (1): 83–90.
Article history
Received:
April 08 2009
Revision Received:
May 07 2009
Accepted:
June 02 2009
Accepted Manuscript online:
June 02 2009
Citation
Oliver Schieweck, Markus Damme, Bernd Schröder, Andrej Hasilik, Bernhard Schmidt, Torben Lübke; NCU-G1 is a highly glycosylated integral membrane protein of the lysosome. Biochem J 15 August 2009; 422 (1): 83–90. doi: https://doi.org/10.1042/BJ20090567
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