Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis

Abstract

Quantitative analysis of tandem-affinity purified cross-linked (x) protein complexes (QTAX) is a powerful technique for the identification of protein interactions, including weak and/or transient components. Here, we apply a QTAX-based tag-team mass spectrometry strategy coupled with protein network analysis to acquire a comprehensive and detailed assessment of the protein interaction network of the yeast 26S proteasome. We have determined that the proteasome network is composed of at least 471 proteins, significantly more than the total number of proteins identified by previous reports using proteasome subunits as baits. Validation of the selected proteasome-interacting proteins by reverse copurification and immunoblotting experiments with and without cross-linking, further demonstrates the power of the QTAX strategy for capturing protein interactions of all natures. In addition, >80% of the identified interactions have been confirmed by existing data using protein network analysis. Moreover, evidence obtained through network analysis links the proteasome to protein complexes associated with diverse cellular functions. This work presents the most complete analysis of the proteasome interaction network to date, providing an inclusive set of physical interaction data consistent with physiological roles for the proteasome that have been suggested primarily through genetic analyses. Moreover, the methodology described here is a general proteomic tool for the comprehensive study of protein interaction networks.

Fig. 1.

Identifying PIPs using a QTAX-based tag-team strategy. (A) Illustration of the yeast 26S proteasome. Subunits in red (Rpn11, Rpn1, Rpn10, and Rpt5) were chosen for HBH tagging. (B) Comparison of the number of PIPs (SILAC ratios >1.5) captured by each bait.

Fig. 2.

(A) Representative MS spectra of tryptic peptides matched to the four known receptor proteins. (i) MH₃³⁺ 617.98, TKVTEPPIAPESATTPGR, matched to Rad23. (ii) MH₂²⁺ 869.93, ATQGFSGADLLYIVQR, matched to Cdc48. (iii) MH₂²⁺ 760.82, QLNDmGFFDFDR, matched to Dsk2. (iv) MH₃³⁺ 617.98, SFQEGLPAPTSVTTSSDKPLTPTK, matched to Ddi1. (B) TOF MS spectra of peptides matched to the PIPs with SILAC ratios as high: (i) MH₃³⁺ 697.97, VAVEFFDDQGDDYNSSKR, matched to Bub3; (ii) MH₃³⁺ 777.02, LAAAQQQAQASGIMPSNEDVATK, matched to Egd2; (iii) MH₃³⁺ 882.04, THSGPTTASNPAPSSTNSSSAPSATNSK, matched to Sok1; and (iv) MH₂²⁺ 761.43, LFDNNLPYLVSVK, matched to Hsm3. ·, light labeled peptide; o, heavy labeled peptide.

Fig. 3.

Validation of the selected PIPs using reciprocal CoIP. (A) I. Western blot of native affinity purified samples using anti-Rpt1 from untagged strain (negative control); Cct4-TAP (L/H 5.6); Hef3-TAP (L/H 5.5); Rvb2-TAP (L/H 9.9); Sec26-TAP (L/H 5.8); Uba1-TAP (L/H 4.6); Vma13-TAP (L/H 3.2); Rpn11-TAP (positive control, 1/20 diluted sample loaded). L/H values are the SILAC ratios determined experimentally using Rpn11. II. Comparison of the lysate input used for purification shown in I. (B) Western blot of the same IP samples used in A (I) from untagged and Hef3-TAP strains using anti-Rpt5 antibody. (C) Validation of Sok1 interaction using Sok1-TAP strain for native purification and Sok1-HBH strain for denaturing purification after in vivo formaldehyde cross-linking. The Western blot was obtained using anti-Rpt5.

Fig. 4.

The interaction map of yeast 26S proteasome with the identified GO complexes (Table S4). The center is the proteasome assembly, and each GO complex is represented as a node. The node size increases as the number of proteins in the complex increase. For nonproteasome complexes, we predict that complexes closer to the center of the figure have a more direct interaction with the proteasome. These data are based on the shortest paths along the interactions in the protein interaction network shown in Fig. S2. The gray color denotes the percentage of proteins of a complex not captured by our study. Interactions between complexes with higher weights (i.e., with more interacting protein pairs among them) are represented with thicker lines. Complex 32 is connected to the proteasome via an identified PIP that is not a member of any GO complex.