Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Int J Mol Sci. 2023 Oct 10;24(20):15036. doi: 10.3390/ijms242015036.
The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function. Here, we show that the IDPR in unconjugated Atg12 is positioned in proximity to the UBL domain, a configuration that is important for the functional structure of the protein. A major deletion in the IDPR disrupts intactness of the UBL domain at the unconjugated C terminus, and a mutation in the predicted α0 helix in the IDPR prevents Atg12 from binding to Atg7 and Atg10, which ultimately affects the protein function in the ubiquitin-like conjugation cascade. These findings provide evidence that the IDPR is an indispensable part of the Atg12 protein from yeast.
酵母中的 Atg12 蛋白是高度保守的泛素样连接系统中不可缺少的多肽，该系统在巨自噬/自噬途径中起作用。Atg12 通过 Atg7 和 Atg10 的作用与 Atg5 共价连接；Atg12-Atg5 缀合物与 Atg16 结合形成 E3 连接酶，该酶在涉及 Atg8 的单独缀合途径中起作用。Atg12 由泛素样 (UBL) 结构域组成，其 N 端之前是一个固有无序的蛋白质区域 (IDPR)，该区域包含蛋白质的大部分，但在其构象和功能上仍难以捉摸。在这里，我们表明未缀合的 Atg12 中的 IDPR 与 UBL 结构域接近，这种构象对于蛋白质的功能结构很重要。IDPR 中的主要缺失会破坏未缀合的 C 末端 UBL 结构域的完整性，并且 IDPR 中预测的α0 螺旋上的突变会阻止 Atg12 与 Atg7 和 Atg10 结合，这最终会影响泛素样缀合级联中的蛋白质功能。这些发现提供了证据，证明 IDPR 是酵母中 Atg12 蛋白不可或缺的一部分。
