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tosis in mast cells and IgE-mediated anaphylaxis. In mouse mast cells, Rab44 is expressed as two isoforms, namely, the long and short forms; however, the characteristics of these two isoforms remain unknown. Here, we investigated secretion and localization of the human long Rab44 isoform and the two mouse isoforms and their mutants expressed in rat basophilic leukemia (RBL)-2H3 cells. Expression of the human long isoform and both mouse isoforms caused an increase in β-hexosaminidase secretion. Confocal and quantitative analyses showed that both human and mouse long isoforms localized mainly to lysosomes while the mouse short isoform localized mainly to the ER. Live imaging with LysoTracker indicated that the size and number of LysoTracker-positive vesicles were altered by the various mutants. Ionomycin treatment partially altered localization of both long isoforms to the plasma membrane and cytosol, whereas it had little effect on colocalization of the short isoform with lysosomes. Mechanistically, both human and mouse Rab44 proteins interacted with vesicle-associated membrane protein 8 (VAMP8), a v-SNARE protein. Therefore, Rab44 isoforms similarly promote lysosomal exocytosis, but exhibit differential localization in mast cells.