Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA; Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.
J Biol Chem. 2021 Jan-Jun;296:100660. doi: 10.1016/j.jbc.2021.100660. Epub 2021 Apr 14.
The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasome-activating nucleotidase proline residue (P91) forms cis- and trans-peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5-P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2-P103A rpt5-P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperone-mediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.
蛋白酶体是一种大型的蛋白酶复合物，可降解许多不同的细胞蛋白质。在真核生物中，26S 蛋白酶体包含六种不同的 ATP 酶相关的细胞活动家族的亚基，Rpt1-Rpt6，它们形成六聚体环作为基底亚基复合物的一部分，驱动底物的展开和易位进入蛋白酶体核心。古菌蛋白酶体仅包含一种与多种细胞活动相关的 Rpt 样 ATP 酶，即蛋白酶体激活核苷酸酶，它形成三聚体二聚体。关键的蛋白酶体激活核苷酸酶脯氨酸残基（P91）在环周围的连续亚基中形成顺式和反式肽键，允许通过上游卷曲螺旋有效地二聚化。然而，对于真核蛋白酶体组装，等效的 Rpt 脯氨酸的重要性尚不清楚。在这里，我们表明，在高度分化的真核生物中，Rpt2、Rpt3 和 Rpt5 中的等效脯氨酸高度保守，而 Rpt1 中的保守性较弱。尽管在芽殖酵母中，单个 Pro-to-Ala 取代并没有强烈地影响生长，但 rpt5-P76A 突变降低了蛋白质水平，并诱导了轻微的蛋白酶体组装缺陷。此外，rpt2-P103A、rpt3-P93A 和 rpt5-P76A 突变与特定蛋白酶体基底组装伴侣缺失组合时都会导致合成缺陷。rpt2-P103A rpt5-P76A 双突变体具有独特的强生长缺陷，归因于蛋白酶体基底形成缺陷。该突变体中的几个 Rpt 亚基形成聚集体，至少部分通过 Hsp42 伴侣介导的蛋白质质量控制被清除。我们提出，保守的 Rpt 连接肽脯氨酸通过促进特定的 ATP 酶异二聚化，促进 26S 蛋白酶体基底的有效组装。
