Burton Randall E, Baker Tania A, Sauer Robert T
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Nat Struct Mol Biol. 2005 Mar;12(3):245-51. doi: 10.1038/nsmb898. Epub 2005 Feb 6.
ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.
ATP 依赖的蛋白质降解主要由底物识别控制。AAA+ HslU ATP 酶被认为可结合蛋白质底物，使其变性，并将未折叠的多肽转运至 HslV 肽酶中。缺乏适用于 HslUV（ClpYQ）的表现良好的高亲和力底物阻碍了对底物结合规则和机制的理解。我们发现 HslUV 能有效降解 Arc 阻遏蛋白，尤其是在热休克温度下。降解取决于 Arc N 端附近的序列。融合蛋白和肽结合实验表明，该序列是一个直接与 HslU 结合的降解标签。此标签与酶的强结合需要 ATP 和 Mg(2+)。此外，将该序列与具有显著机械稳定性的蛋白质融合会导致其完全降解。因此，这些实验表明 HslUV 是一种强大的蛋白质解折叠酶，并且 HslU ATP 酶对底物的初始结合必定发生在 ATP 结合之后。
