Identification of a lysosomal protein causing lipid transfer, using a fluorescence assay designed to monitor membrane fusion between rat liver endosomes and lysosomes.
In the present and previous studies [Mullock, Perez, Kuwana, Gray and Luzio (1994) J. Cell Biol. 126, 1173-1182], we have attempted to investigate endosome-lysosome fusion using an assay based on the dilution of the self-quenching fluorescent lipid probe octadecylrhodamine. Although some characteristics of fluorescence dequenching were consistent with those observed in other cell-free assays, we have now demonstrated that increased fluorescence was due to leakage of an intralysosomal lipid-transfer protein. This protein was purified and found to be a 22 kDa molecule with sequence, immunological and functional characteristics strongly suggesting that it is the rat homologue of human GM2-activator protein. Both the 22 kDa protein and recombinant human GM2-activator protein caused fluorescence dequenching either when mixed with octadecylrhodamine-loaded endosomes and lysosomal membranes or in a liposome system. The data were consistent with GM2-activator protein acting as an octadecylrhodamine-transfer protein. Antibodies to the 22 kDa protein added to cell-free endosome-lysosome content-mixing assays had no effect, although they could inhibit fluorescence dequenching caused by the protein. Thus this protein is not required in any fusion event involved in delivery of ligands from endosomes to lysosomes. The existence within an intracellular organelle of a protein capable of acting as an octadecylrhodamine-transfer protein suggests the need for caution in the interpretation of fluorescence-dequenching assays using mammalian subcellular fractions.
Kuwana
T
T
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Mullock
B M
BM
Luzio
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D005453:Fluorescence; D049289:G(M2) Activator Protein; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012235:Rhodamines / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D002352:Carrier Proteins; D049289:G(M2) Activator Protein; D008081:Liposomes; D011506:Proteins; D012235:Rhodamines; C030363:lipid transfer protein; C044542:octadecyl Rhodamine B chloride