Expanded Interactome of the Intrinsically Disordered Protein Dss1

Abstract

Dss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome.

Keywords: PCI domain; intrinsically disordered proteins; proteasome; protein degradation.

Graphical abstract

Figure 1

The C-Terminal Region of Dss1 Forms a Dynamic Intramolecular Interaction Limiting Binding Site Access (A) Dss1 consists of extended structures, including the binding sites BS-I and -II and the transient α helix in the C terminus. (B) The presence of ¹⁴N Dss1-N71C-MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) had no effect on the peak intensities of ¹⁵N-Dss1-WT, indicating no inter-molecular interaction. (C) Intramolecular effect from internal MTSL labeling measured from the effects on the transversal relaxation rate of backbone amide protons of Dss1-N71C-MTSL, resulting in a decrease in the intensity (height) of heteronuclear single quantum coherence (HSQC) peaks of the paramagnetic sample compared to the intensity of the HSQC peaks without the MTSL label. (D) Truncation of the helix in Dss1Δhelix had no long-range effect on the amide chemical shifts in Dss1. (E) Different possible conformers of Dss1. The transient α helix in Dss1 forms a dynamic interaction with residues of BS-I, representing a conformational ensemble shielding the BS-I. See also Figure S1.

Figure 2

Quantitative Mass Spectrometry and Dss1 Binding Partners (A) Dss1 binding partners were clustered into known protein complexes. The PCI-domain-containing complexes are framed (transparent). All data are included in Data S1. (B) Extracts from wild-type strains, expressing the indicated 6His-tagged proteins, were used for co-precipitation with GST-Dss1 and GST. The precipitated material was analyzed by blotting for the 6His tag. Equal loading was checked by staining with Coomassie brilliant blue (CBB). (C) Cells with HA-tagged Csn1 and expressing either GFP or GFP-Dss1 were used for immunoprecipitation (IP) using GFP-trap. The precipitated material was analyzed by blotting for the HA-tag or GFP (Dss1). (D) Plot of the fold change in GFP-Dss1 versus GFP (x axis) versus the fold change in GFP-Dss1Δhelix versus GFP (y axis). Proteins marked in green were unaffected by deletion of the C-terminal region. Proteins marked in blue were more associated with Dss1Δhelix, and proteins marked in yellow were less associated with Dss1Δhelix. See also Figures S2 and S3.

Figure 3

The C-Terminal Region Blocks Dss1 Binding to ACLY (A) dss1Δ transformed to produce GFP, GFP-Dss1, and GFP-Dss1Δhelix was used for IP using GFP-trap. The precipitated material was analyzed by SDS-PAGE and CBB staining. The identity of Acl1 and Acl2 was determined by mass spectrometry. (B) dss1Δ cells, expressing the Dss1 variants (upper panel), were used for IP using GFP-trap. BS-I^∗ and BS-II^∗ indicate point mutations in the BS-I and BS-II binding sites (BS-I^∗: L40A/W41A/W45A; BS-II^∗: F18A/F21A/W26A). The precipitated material was analyzed by blotting. (C) The indicated strains expressing GFP-Dss1Δhelix were used for IP using GFP-trap. The precipitated material was analyzed by SDS-PAGE and CBB staining. (D) The solubility of 6His-tagged Acl1 was determined by centrifugation of whole-cell extracts and blotting. (E) Blots as shown in (D) were quantified by densitometry and presented as percent of total (sol. + insol.). The error bars indicate the SEM (n = 3). See also Figure S4.

Figure 4

The C-Terminal Region Is Required for Dss1 Interaction with Mitotic Septins (A) Cells with HA-tagged Spn3 or Spn4 and expressing either GFP or GFP-Dss1 were used for IP using GFP-trap. The precipitated material was analyzed by blotting for the HA-tag or GFP. (B) Cells with HA-tagged Spn3 and expressing either GFP or GFP-Dss1 variants (upper panel) were used for IP using GFP-trap. BS-I^∗ and BS-II^∗ indicate point mutations in the BS-I and BS-II binding sites (BS-I^∗: L40A/W41A/W45A; BS-II^∗: F18A/F21A/W26A). The precipitated material was analyzed by SDS-PAGE and blotting for the HA-tag or GFP. (C) The solubility of GFP-tagged Spn3 was determined by centrifugation of whole-cell extracts and blotting. (D) Blots as shown in (C) were quantified by densitometry and presented as percent of total (sol. + insol.). The error bars indicate the SEM (n = 3). (E) The septum ring of wild-type and dss1Δ cells was observed by fluorescence microscopy using Spn3-GFP as a marker. Scale bar represents 5 μm. (F) Wild-type and dss1Δ cells expressing Spn3-GFP were observed at 27°C over time by fluorescence microscopy, and the duration of the GFP signal was quantified. Each circle or square represents the timing in 1 cell. Black bars represent the average and SEM; n > 94 cells; 2 experiments. Unpaired two-tailed t test; ^∗∗∗∗p < 0.0001.