Laboratory of Molecular Biology, NIDDK, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2010 Mar 5;285(10):7794-804. doi: 10.1074/jbc.M109.069088. Epub 2010 Jan 4.
The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.
Hermansky-Pudlak 综合征（HPS）是一种遗传性色素减退和出血性疾病，由溶酶体相关细胞器（LRO）如黑素体和血小板致密体的生物发生缺陷引起。HPS 是由人类 8 个基因和小鼠 16 个基因中的突变引起的。其中两个基因，HPS1 和 HPS4，编码溶酶体相关细胞器生物发生复合物-3（BLOC-3）的组成部分。本文中，我们表明在昆虫细胞中产生的重组 HPS1-HPS4 可以作为 1:1 的异二聚体有效地分离出来。分析超速离心表明，该复合物的分子量为 146 kDa，相当于天然复合物的分子量以及 HPS1 和 HPS4 的预测分子量之和。这表明 HPS1 和 HPS4 在没有任何其他蛋白质的情况下直接相互作用，作为 BLOC-3 的一部分。有限的蛋白水解和缺失分析表明，两个亚基在其大部分长度上相互作用，唯一的例外是 HPS4 中心部分的一个长的无结构环。相互作用筛选显示 BLOC-3 与内体 GTP 酶 Rab9 的 GTP 结合形式具有特异性和强相互作用。这种相互作用由 HPS4 和 Rab9 的开关 I 和 II 区域介导。这些特征表明 BLOC-3 可能在 LRO 的生物发生中作为 Rab9 效应物发挥作用。
