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.

Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

“…A smaller fraction of 5-8 kDa HA-liposomes (Figure 7F) and plain liposomes (Figure 7G) also displayed partial co-localization with the LysoTracker Green after 2 hours of incubation. To further confirm the results, Texas Red labeled 3 kDa dextran, a probe known for labeling the lysosomal compartment, 41 was used as a positive control for lysosomal localization. As expected, Texas Red 3 kDa dextran was completely co-localized with the LysoTracker Green staining (Figure 7H).…”

Proteomics of the lysosome

“…Lysosomal p40 (gene name 4930471M23Rik) was originally identified in a preparation of lysosomal membranes derived from rat liver lysosomes [91]. Lysosomal localization was subsequently demonstrated by cosedimentation with lysosomal marker proteins after subcellular fraction and colocalization GFP-tagged versions of p40 with LAMP-1 [91].…”

“…Lysosomal localization was subsequently demonstrated by cosedimentation with lysosomal marker proteins after subcellular fraction and colocalization GFP-tagged versions of p40 with LAMP-1 [91]. Mouse p40 is a highly hydrophobic integral membrane protein that has between seven to ten transmembrane domains that are linked to each other by relatively short loops.…”

“…Unlike most lysosomal membrane proteins, p40 does not contain any N-linked carbohydrate residues. The lysosomal half-life of p40 was estimated ~10 h [91] which is comparable with highly-glycosylated proteins such as LAMP-1 or LIMP-1, -2 or -3 for which deglycosylation results in an increased proteolysis [92, 93]. Sequence homologies suggest that p40 may function as a lysosomal nucleotide-sugar transporter.…”

Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes

“…In particular, treatment of rodents with Triton WR-1339 results in a marked decrease in the buoyant density of liver lysosomes (converting them to "tritosomes") but not of other organelles (11). This density shift is a specific hallmark of lysosomal proteins that has been used in the case-by-case verification of lysosomal candidates identified in proteomics experiments (12)(13)(14)(15)(16)(17).…”