NCU-G1 is a highly glycosylated integral membrane protein of the lysosome.
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.
Schieweck
Oliver
O
Abteilung Biochemie II, Georg-August-Universität Göttingen, Göttingen, Germany.
Damme
Markus
M
Schröder
Bernd
B
Hasilik
Andrej
A
Schmidt
Bernhard
B
Lübke
Torben
T
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D019295:Computational Biology; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006031:Glycosylation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D050505:Mutant Proteins; C543310:NCU-G1 protein, mouse; D014157:Transcription Factors; D011092:Polyethylene Glycols; C016811:tyloxapol