IDENTIFICATION OF A LYSOSOMAL PROTEIN CAUSING LIPID TRANSFER, USING AFLUORESCENCE 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, 1273-1182], we have attempted to inves tigate endosom-lysosome fusion using an assay based on the dilution of the self-quenching fluorescent lipid probe octadecylrhodamine. Althou gh some characteristics of fluorescence dequenching were consistent wi th those observed in other cell-free assays, we have now demonstrated that increased fluorescence was due to leakage of an intralysosomal li pid-transfer protein. This protein was purified and found to be a 22 k Da molecule with sequence, immunological and functional characteristic s strongly suggesting that it is the rat homologue of human G(M2)-acti vator protein. Both the 22 kDa protein and recombinant human G(M2)-act ivator protein caused fluorescence dequenching either when mixed with octadecylrhodamine-loaded endosomes and lysosomal membranes or in a li posome system. The data were consistent with G(M2)-activator protein a cting 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 c aused 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 a cting as an octadecylrhodamine-transfer protein suggests the need for caution in the interpretation of fluorescence-dequenching assays using mammalian subcellular fractions.