Tabata Hiroyuki, Kawamura Nobuyuki, Sun-Wada Ge-Hong, Wada Yoh
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyoto, Japan.
Cell Tissue Res. 2008 Jun;332(3):447-60. doi: 10.1007/s00441-008-0597-5. Epub 2008 Apr 12.
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.
黑素小体是一种专门用于黑色素合成的细胞器，是溶酶体相关细胞器之一。据报道，其内腔通过液泡型H(+)-ATP酶（V-ATP酶）酸化。哺乳动物的V-ATP酶在其亚基异构体中表现出结构多样性；就膜内在亚基a而言，已发现四种异构体（a1-a4）定位于不同的亚细胞区室。在本研究中，我们表明a3异构体与小鼠黑素细胞中的黑素小体标记蛋白Pmel17共定位。嗜酸性探针（LysoSensor和DAMP）积聚在无色素的Pmel17阳性黑素小体中，并且DAMP的积聚对V-ATP酶的特异性抑制剂巴弗洛霉素A1敏感。然而，亚基a的异构体均不与高度色素沉着的成熟黑素小体相关，在成熟黑素小体中嗜酸性探针也不积聚。a3基因座发生无效突变的oc/oc小鼠在黑色素生成方面没有明显缺陷。在突变的黑素细胞中，a2异构体的表达适度升高，并且该异构体的相当一部分定位于早熟黑素小体。这些观察结果表明，V-ATP酶使早熟黑素小体的内腔保持酸性，而黑素小体酸化在成熟黑素小体中不太明显。
