Institute of Genetics, Heinrich-Heine-Universitaet Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany.
Cells. 2024 Jul 10;13(14):1174. doi: 10.3390/cells13141174.
Members of the LGD/CC2D1 protein family contain repeats of the family-defining DM14 domains. Via this domain, they interact with members of the CHMP family, which are essential for the ESCRT machinery-mediated formation of intraluminal vesicles during endosome maturation. Here, we investigate the requirement of the DM14 domains for the function of Lgd in detail. We found that although both odd-numbered DM14s can act in a functionally redundant manner, the redundancy is not complete and both contribute to the full function of Lgd. Our analysis indicates that some of the AAs that form the KARRxxR motif of the onDM14s are not exchangeable by similarly charged AAs without loss of function, indicating that they not only provide charge, but also fulfil structural roles. Furthermore, we show that the region of Lgd between DM14-4 and the C2 domain as well as its C-terminal region to the C2 domain are important for protein stability/function. Moreover, we analysed the importance of AAs that are conserved in all DM14 domains. Finally, our analysis of the ortholog of Lgd revealed that it has only one DM14 domain that is functionally equivalent to the onDM14s. Altogether, the results further the understanding of how Lgd family members regulate the ESCRT machinery.
LGD/CC2D1 蛋白家族的成员包含家族定义的 DM14 结构域的重复序列。通过这个结构域，它们与 CHMP 家族的成员相互作用，后者对于内体成熟过程中 ESCRT 机器介导的腔内小泡形成至关重要。在这里，我们详细研究了 DM14 结构域对 Lgd 功能的要求。我们发现，尽管奇数 DM14 都可以以功能上冗余的方式发挥作用，但冗余并不完全，两者都有助于 Lgd 的全部功能。我们的分析表明，形成奇数 DM14s 的 KARRxxR 基序的一些氨基酸不能被同样带电荷的氨基酸取代而不失功能，这表明它们不仅提供电荷，而且还发挥结构作用。此外，我们还表明，DM14-4 和 C2 结构域之间的 Lgd 区域以及其 C 末端到 C2 结构域的区域对于蛋白质稳定性/功能很重要。此外，我们分析了所有 DM14 结构域中保守的氨基酸的重要性。最后，我们对 Lgd 的同源物的分析表明，它只有一个 DM14 结构域，与奇数 DM14s 具有相同的功能。总之，这些结果进一步了解了 Lgd 家族成员如何调节 ESCRT 机器。
