Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, United States.
Department of Chemistry, Dartmouth College, Hanover, NH 03755, United States.
J Mol Biol. 2021 Mar 5;433(5):166809. doi: 10.1016/j.jmb.2021.166809. Epub 2021 Jan 21.
Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.
自噬是真核细胞中的一种批量降解机制。酵母中这个过程的一个基本步骤，即 Atg8 的脂质化，效率依赖于 Atg16 的存在，Atg16 是 Atg12-Atg5-Atg16 复合物的一个亚基，作为泛素样反应中的 E3 样酶。目前对酿酒酵母 S. cerevisiae 中 Atg16 的功能结构的看法来自两个晶体结构，这些结构揭示了 Atg5 相互作用的α-螺旋通过柔性接头与 Atg16 的另一个α-螺旋相连，然后组装成同源二聚体。这种观点并不能解释之前的体外研究结果，这些结果表明，在添加 Atg16 后，Atg12-Atg5 缀合物依赖于 Atg16 发生膜变形和与脂质体结合。在这里，我们表明 Atg16 既是同源二聚体形成蛋白又是外周膜结合蛋白。这两个特征是由新生蛋白中两个不同的无序区域所决定的。Atg16 在体内通过具有卷曲螺旋样倾向和强烈插入膜的疏水面的两性α-螺旋（氨基酸残基 113-131）与膜结合。另一个蛋白质区域（残基 64-99）具有卷曲螺旋倾向，但没有两性，对于 Atg16 膜锚定是可有可无的。该区域作为形成 Atg16 同源二聚体所必需的亮氨酸拉链。这两个不同区域中的任何一个突变都会导致 Atg16 蛋白完全不能挽救 atg16Δ 细胞的自噬缺陷表型，而野生型则可以。总的来说，这项研究的结果提供了一个自噬体形成的分子机制模型。
