NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

Oliver Schieweck , Markus Damme , Bernd Schröder , Andrej Hasilik , Bernhard Schmidt , Torben Lübke (1)

1 Biochemie II

Until recently, a modest number of approximately 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. Here, we demonstrate the lysosomal localization of the mouse ortholog of the human C1orf85 protein that had been termed kidney predominant protein NCU-G1 (Acc.No: 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 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 for the tyrosine residue within the putative sorting motif.

Mots clés: Life Sciences

Oliver Schieweck, Markus Damme, Bernd Schröder, Andrej Hasilik, Bernhard Schmidt, et al.. NCU-G1 is a highly glycosylated integral membrane protein of the lysosome. Biochemical Journal, 2009, 422 (1), pp.83-90. ⟨10.1042/BJ20090567⟩. ⟨hal-00479186⟩