Sem1/DSS1 accelerates ATP-dependent substrate unfolding by the proteasome through a conformation-dependent intercomplex contact

Abstract

The 26S proteasome is an ~70 subunit ATP-dependent chambered protease that destroys proteins via multiple highly coordinated processing steps. The smallest and only intrinsically disordered proteasome subunit, Sem1 (DSS1 in metazoans), is critical for efficient substrate degradation despite lacking obvious enzymatic activities and being located far away from the proteasome's catalytic centers. Dissecting its role in proteolysis using cell-based approaches has been challenging because Sem1 also controls proteasome function indirectly via its role in proteasome biogenesis. To circumvent this challenge, we reconstituted Sem1-deficient proteasomes in vitro from purified components and systematically dissected its impact on distinct processing steps. Whereas most substrate processing steps are independent of Sem1, ATP-dependent unfolding is stimulated several-fold. Using structure-guided mutagenesis and engineered protein crosslinking, we demonstrate that Sem1 allosterically regulates ATP-dependent substrate unfolding via a distal conformation-dependent intersubunit contact. Together, this work reveals how a small, unstructured subunit comprising < 0.4% the total size of the proteasome can augment substrate processing from afar, and reveals a new allosteric pathway in controlling proteolysis.

CRISPR/Cas9-Targeted Disruption of Two Highly Homologous Arabidopsis thaliana DSS1 Genes with Roles in Development and the Oxidative Stress Response

“…In order to show that mutant lines are T-DNA free, i.e., that they do not contain CRISPR/Cas9 cassette between T-DNA right/left border sequence, we performed PCR on gDNA isolated from dss1(I). 19 and dss1(V).20 and primers that amplify 423 bp of U6-26p promoter sequence. The corresponding gel electrophoresis is presented as Figure S3 in the Supplementary material.…”

“…It contributes to the stability of the 19S proteasome lid by the successive recruitment of the regulatory particle non-ATPase 3 (Rpn3) and Rpn7 lid subunits [8,17,18]. While proteasome assembly is possible, it is not fully functional in the absence of Sem1 because Sem1 is necessary for Rpn7 modulation for efficient ATP-dependent substrate unfolding during proteolysis [19].…”

Assessing Transcriptomic Responses to Oxidative Stress: Contrasting Wild-Type Arabidopsis Seedlings with dss1(I) and dss1(V) Gene Knockout Mutants

“…As the smallest regulatory and structural subunit of the 26S proteasome, besides ubiquitin-binding activity, Dss1 also participates in the correct proteasome assembly. Dss1 contributes to the stability of the proteasome by effectively recruiting its subunits Rpn3 and Rpn7, which normally have a low affinity for each other [ 23 , 25 , 26 ]. Although complete assembly of the proteasome is possible in the absence of Dss1, the proteolytic activity of this proteasome is incomplete.…”

“…Although complete assembly of the proteasome is possible in the absence of Dss1, the proteolytic activity of this proteasome is incomplete. During proteolysis, Dss1 is necessary for the fine modulation of Rpn7 in the process of efficient ATP-dependent substrate unwinding [ 25 ].…”

Phosphorylation of Schizosaccharomyces pombe Dss1 mediates direct binding to the ubiquitin‐ligase Dma1 in vitro

“…Deleted in split hand/split foot 1 (Dss1—Sem1 in budding yeast) is a small, eukaryotic, conserved, and intrinsically disordered protein (IDP) that serves as a subunit of the large 26S proteasome complex (Funakoshi et al, 2004 ), where it is important for its assembly (Jossé et al, 2006 ; Kolog Gulko et al, 2018 ; Tomko & Hochstrasser, 2014 ) and allosteric regulation of substrate unfolding (Reed et al, 2022 ). Yeast mutants lacking Dss1 display a temperature‐dependent growth defect largely linked to faulty proteasome assembly (Tomko & Hochstrasser, 2014 ).…”