BIOGENESIS OF MELANOSOMES

(改编自申请者的摘要)-这是一个长期目标 建议的研究是为了阐明皮肤的分子基础 黑化以了解人类疾病的发病机制 影响色素沉着并设计治疗方法。通过组合 利用鼠标的力量进行细胞和分子生物学技术 遗传学，黑素细胞产生其基因的基本机制 专门的细胞器，黑素小体，将通过三个 未来5年的具体目标：(1)阐明 黑素小体。已知和新发现的黑素小体的分布 我们将研究蛋白质与其他亚细胞蛋白质的关系。 认为黑素小体是通过两部分途径构建的假说 其中平滑的内质网来源的前黑素小体与高尔基体来源的融合 将对水泡进行测试。免疫显微镜，亚细胞分离， 代谢标记和转基因将被用来评估如何 个别蛋白质到达黑素小体。通过将这些组合起来 用Agti信号蛋白处理黑素细胞的技术， 将生产从欧盟转移到菲黑素小体，洞察它 希望能深入了解两者之间的根本区别 两种类型的黑素小体；(2)探讨两者之间的关系 黑素小体和“分泌型溶酶体”。黑素小体是 细胞器的溶酶体谱系，与 “分泌型溶酶体。”黑素小体是否包含了所有溶酶体 我们将研究黑素细胞的功能。潜在的联系 将确定AP-3接头复合体与黑素体的比例，也将确定 它与黑素体膜胞浆尾巴的相互作用 蛋白质；(3)buff(Bf)基因座和65 kDa黑素小体的作用 蛋白。黑素细胞和黑素细胞中65 kDa外周膜蛋白 含有“分泌型溶酶体”的细胞已被鉴定为 定位于黑素小体的胞浆表面。这种蛋白质的水平 是由buff(Bf)和beige(Bg)基因座的突变而改变的，这两个基因都是已知的 从而影响分泌型溶酶体途径。蛋白质将会是 测序并确定其与BG基因产物的关系。这个 将克隆编码该蛋白质的cdna，如果它不是剪接形式 在BG中，它的染色体位置将被定位在小鼠身上。如果它映射到 对于bf或其他毛色基因，将使用突变分析。 以确定它是否在该位置编码。蛋白质的合成， 分布，以及它与其他蛋白质的相互作用将被研究 以阐明其功能。

(Adapted from the applicant's abstract)- The long-term goal of this proposed research is to elucidate the molecular basis of cutaneous melanization in order to understand the pathogenesis of human diseases affecting pigmentation and to devise treatments for them. By combining techniques of cellular and molecular biology with the power of mouse genetics, the basic mechanisms by which a melanocyte creates its specialized organelle, the melanosome, will be addressed through three specific aims over the next 5 years: (1) Elucidate the biogenesis of melanosomes. The distribution of known and newly identified melanosomal proteins will be examined in relation to other subcellular proteins. The hypothesis that melanosomes are constructed via a bipartite pathway wherein smooth ER-derived premelanosomes fuse with Golgi-derived vesicles will be tested. Immunomicroscopy, subcellular fractionation, metabolic labeling, and transfection will be used to assess how individual proteins arrive at melanosomes. By combining these techniques with the treatment of melanocytes with Agouti signal protein, which shifts production from the eu- to the pheo-melanosome, insight it is hoped will be gained into the fundamental differences between these two types of melanosomes; (2) explore the relationship between melanosomes and "secretory lysosomes." The melanosome is a member of the lysosomal lineage of organelles which most closely resembles the "secretory lysosome." Whether melanosomes have subsumed all lysosomal function in melanocytes will be investigated. The potential association of the AP-3 adaptor complex with melanosomes will be determined, as will its interaction with the cytosolic tails of melanosomal membrane proteins; (3) role of the buff (bf) locus and 65 kDa melanosomal protein. A 65 kDa peripheral membrane protein in melanocytes and in cells containing "secretory lysosomes" has been identified which localizes to the cytosolic face of melanosomes. Levels of this protein are altered by mutation at the buff (bf) and beige (bg) loci, both known to affect the secretory lysosomal pathway. The protein will be sequenced and its relationship to the bg gene product determined. The cDNA encoding the protein will be cloned and, if it is not a splice form of bg, its chromosomal location will be mapped in the mouse. If it maps to bf or to another coat color locus, mutational analysis will be used to determine if it is encoded at that locus. The protein's synthesis, distribution, and its interactions with other proteins will be studied to elucidate its function.