The two-domain architecture of LAMP2A within the lysosomal lumen regulates its interaction with HSPA8/Hsc70
The two-domain architecture of LAMP2A within the lysosomal lumen regulates its interaction with HSPA8/Hsc70
复制标题
溶酶体腔内 LAMP2A 的两域结构调节其与 HSPA8/Hsc70 的相互作用
DOI：
10.1080/27694127.2022.2069968
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Hara-Yokoyama Miki
中科院分区：
文献类型：
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作者：
Ikami Yuta;Terasawa Kazue;Watabe Tetsuro;Yokoyama Shigeyuki;Hara-Yokoyama Miki
分子伴侣介导的自噬（Chaperone-mediated autophagy，CMA）是一种独特的蛋白水解途径，由热休克蛋白8（HSPA 8/Hsc 70，heat shock protein 8）识别的胞浆蛋白被转运到溶酶体中降解。LAMP 2A（溶酶体相关膜蛋白2A）将底物/伴侣复合物募集至溶酶体膜。LAMP 2A的结构包括由两个同源亚结构域（N结构域和C结构域，均具有β-棱柱折叠）组成的大内腔结构域、跨膜结构域和短胞质尾。虽然已经提出了LAMP 2A分子之间的亲嗜性相互作用以及HSPA 8和LAMP 2A之间的相互作用，但是这些相互作用是直接的还是由其他分子介导的仍有待阐明。我们研究了使用扩展的遗传密码技术，产生光交联和/或空间位阻在哺乳动物细胞中的指定接口的相互作用。结果表明，LAMP 2A分子通过在其C-结构域中以β-棱柱的一侧彼此面对而组装。我们还检测到LAMP 2A和HSPA 8的胞质尾区之间的光交联，揭示了这种直接的相互作用。我们发现N-结构域的截短减少了与LAMP 2A共免疫沉淀的HSPA 8的量。我们目前的研究结果表明，两个结构域的内腔结构域的LAMP 2A的基础上与HSPA 8在溶酶体的细胞质表面的相互作用。
Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by HSPA8/Hsc70 (heat shock protein 8) are transported into lysosomes for degradation. LAMP2A (lysosomal-associated membrane protein 2A) recruits the substrate/chaperone complex to the lysosome membrane. The structure of LAMP2A comprises a large lumenal domain composed of two homologous subdomains (N-domain and C-domain, both with the β-prism fold), a transmembrane domain, and a short cytoplasmic tail. Although the homophilic interaction between LAMP2A molecules and the interaction between HSPA8 and LAMP2A have been suggested, whether the interactions are direct or mediated by other molecules has remained to be elucidated. We investigated the interactions by using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces in mammalian cells. The results suggested that LAMP2A molecules assemble by facing each other with one side of the β-prism in their C-domains. We also detected the photo-crosslinking between the cytoplasmic tail of LAMP2A and HSPA8, revealing this direct interaction. We found that the truncation of the N-domain reduced the amount of HSPA8 that coimmunoprecipitates with LAMP2A. Our present results suggest that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with HSPA8 at the cytoplasmic surface of the lysosome.