Rab7 regulates phagosome maturation in Dictyostelium

A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis. Since R ab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation. Expressi on of GFP-Rab7 resulted in labeling of both early and late phagosomes conta ining yeast, but not forming phagocytic cups. In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal sy stem and maturing phagosomes, latex bead containing (LBC) phagosomes were p urified from wild-type cells at various times after internalization. Glycos idases, cysteine proteinases, Rab7 and lysosomally associated membrane prot eins were delivered rapidly to nascent phagosomes in control cells. LBC pha gosomes isolated from cells overexpressing dominant negative (DN) Rab7 cont ained very low levels of LmpA (lysosomal integral membrane protein) and alp ha -mannosidase was not detectable. Interestingly, cysteine proteinases wer e delivered to phagosomes as apparent proforms in cells overexpressing DN R ab7. Despite these defects, phagosomes in cells overexpressing DN Rab7 matu red to form multi-particle spacious phagosomes, except that these phagosome s remained significantly more acidic than control phagosomes. These results suggested that Rab7 regulates both an early and late steps of phagosomal m aturation, similar to its role in the endo/lysosomal system.