title: Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes.
abstract: 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 beta-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.
authors.affiliation: URPHIM, Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, B-5000 Namur, Belgium.
authors.forename: Marielle
authors.initials: M
authors.lastname: Boonen
authors.forename: Isabelle
authors.initials: I
authors.lastname: Hamer
authors.forename: Muriel
authors.initials: M
authors.lastname: Boussac
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Delsaute
authors.forename: Bruno
authors.initials: B
authors.lastname: Flamion
authors.forename: Jérôme
authors.initials: J
authors.lastname: Garin
authors.forename: Michel
authors.initials: M
authors.lastname: Jadot
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; C018589:lysosomal proteins