Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

Inactivation of gamma-cystathionase by beta, beta, beta-trifluoroalanine, a suicide inactivator of the enzyme, results in covalent labeling of an amino group of the protein [Silverman, R. B., & Abeles, R. H. (1977) Biochemistry 16, 5515-5520]. We have established that this modified amino function is the epsilon-NH2 group of a lysine residue. A heptapeptide which includes this modified lysine residue was isolated, and its sequence was found to be Cys-Ser-Ala-Thr-Lys-Tyr-Met. The amino acid sequence was the same as that determined for peptides containing the active-site lysine residue which forms a Schiff base with pyridoxal phosphate. Therefore the epsilon-NH2 group of the active-site lysine which binds pyridoxal phosphate is capable of interacting with the beta carbon of trifluoroalanine, and presumably the beta carbon of normal substrates. We therefore propose that this lysine residue may function as a proton-transfer agent in the reactions catalyzed by gamma-cystathionase.