The nascent phagosome progressively establishes an acidic milieu by acquiring a proton pump, the vacuolar-type ATPase (V-ATPase). However, the origin of phagosomal V-ATPase remains poorly understood. We found that phagosomes were enriched with the V-ATPase a3 subunit, which also accumulated in late endosomes and lysosomes. We modified the mouse Tcirg1 locus encoding subunit a3, to express an a3-GFP fusion protein. Live-cell imaging and immunofluorescence microscopy revealed that nascent phagosomes received the a3-GFP from tubular structures extending from lysosomes located in the perinuclear region. Macrophages from a3-deficient mice exhibited impaired acidification of phagosomes and delayed digestion of bacteria. These results show that lysosomal V-ATPase is recruited directly to the phagosomes via tubular lysosomes to establish the acidic environment hostile to pathogens.
The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H(+)-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1-a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes.
Vacuolar-type proton ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across membranes. Mammalian cells express four isoforms of the a subunit of V-ATPase. Previously, we have shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the membranes of insulin-containing granules in the beta cells. The a3 isoform functions in the regulation of hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in regulated secretion.
BackgroundVacuolar-type proton transporting ATPase (V-ATPase) is involved in the proper development of visual function. Mutations in the Tcirg1 (also known as Atp6V0a3) locus, which encodes the a3 subunit of V-ATPase, cause severe autosomal recessive osteopetrosis (ARO) in humans. ARO is often associated with impaired vision most likely because of nerve compression at the optic canal. We examined the ocular phenotype of mice deficient in Tcirg1 function.Methodology/Principal FindingsX-ray microtomography showed narrowed foramina in the skull, suggesting that optic nerve compression occurred in the a3-deficient (Tcirg1
−/−) mice. The retina of the mutant mice had normal architecture, but the number of apoptotic cells was increased at 2–3 wks after birth. In the ocular system, the a3 subunit accumulated in the choriocapillary meshwork in uveal tissues. Two other subunit isoforms a1 and a2 accumulated in the retinal photoreceptor layer. We found that the a4 subunit, whose expression has previously been shown to be restricted to several transporting epithelia, was enriched in pigmented epithelial cells of the retina and ciliary bodies. The expression of a4 in the uveal tissue was below the level of detection in wild-type mice, but it was increased in the mutant choriocapillary meshwork, suggesting that compensation may have occurred among the a subunit isoforms in the mutant tissues.ConclusionsOur findings suggest that a similar etiology of visual impairment is involved in both humans and mice; thus, a3-deficient mice may provide a suitable model for clinical and diagnostic purposes in cases of ARO.
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