Subunit architecture of multimeric complexes isolated directly from cells

Abstract

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Figure 1

MS of intact dimeric and trimeric complexes. (A) MS of the scavenger decapping complex shows only one principal species, with charge states labelled A18–A21, consistent with a heterodimer of Dcs1 and Dcs2-CBP. Inset: MS/MS spectrum obtained after isolation and dissociation of the 18+ charge state highlighted in blue to yield products B and C (Dcs1 and Dcs2-CBP, respectively). (B) Nuclear cap-binding complex consisting of Mud13-CBP and Sto1 (series E) illustrating interaction with SRP1 (series F). The dashed lines indicate that Srp1 could be associated with either or both of Mud13-CBP and Sto1. CBP, calmodulin-binding peptide; MS, mass spectrometry.

Figure 2

MS of the intact yeast exosome complex isolated with the Csl4-tagged protein. The main panel shows a charge state series labelled A and B for the intact exosome and complex without Csl4, respectively. (A) MS/MS and (B) MS spectra of Rrp41-tagged exosome complex, showing the 85 kDa trimer and dissociation products from its 17+ charge state. The dimer and monomer products indicate that the 85 kDa species correspond to the trimer Rrp46:Rrp45:Rrp40. Charge state labels: C Rrp46, D Rrp40, E Rrp45:Rrp40:Rrp46, F Rrp45:Rrp46, G Rrp45:Rrp40, H Rrp45, J yeast heat-shock protein (SSA1/SSA2). (C) MS of Dis3-tagged and (D) Rrp41-tagged exosome after disruption of the complex in solution using 20% methanol and 33% dimethylsulphoxide, respectively. A heterotetramer K and three different dimers L, M and N are readily identified in the spectra. MS, mass spectrometry.

Figure 3

Subcomplexes obtained after disruption of the exosome complexes in solution showing common dissociation pathways. (A) MS of Dis3-tagged exosome in 20% methanol. Charge states labelled in green are [exo-Csl4-Rrp43] and [exo-Csl4-Rrp43-Mtr3], series D (327 kDa) and C (299 kDa), respectively, where [exo] refers to the intact, ten-component complex, whereas those in blue are assigned as Rrp45:Rrp41:Rrp42:Rrp4:Dis3 with Mtr3 (series B, 276 kDa) and without Mtr3 (series A, 249 kDa). Charge state E39 is from [exo-Csl4]. (B) MS of Rrp41-tagged exosome in 33% dimethylsulphoxide. Charge states labelled in blue are Rrp45:Rrp41:Rrp4:Rrp42:Dis3 with Mtr3 (series H, 277 kDa) and without Mtr3 (series F, 249 kDa). Series G (258 kDa) is [exo-Csl4-Dis3] and J (371 kDa) is [exo-Csl4]. (C) Interaction map derived from MS data. The RNA-binding proteins Rrp40, Csl4 and Rrp4 each contact two ring dimers, whereas Dis3 contacts one or more proteins from the group Rrp45:Rrp41:Rrp42. MS, mass spectrometry.

Figure 4

Summary of the exosome subcomplexes and steps taken to build the 3D model. The ten-protein intact complex is shown in grey. Dotted lines indicate the subset of proteins the interactions of which are unknown at each step. Perturbation in solution was used to generate dimeric and trimeric complexes, confirmed by MS/MS (A), and overlap of these complexes is used to derive their subunit interactions (B). The loss of Mtr3 and Rrp43 orientates the Mtr3:Rrp42 dimer within the ring (C), and solution-phase loss of Rrp43, Mtr3 and Csl4 is used to locate Csl4 and Rrp4 (D,E). Analysis of subcomplexes containing Dis3 positions this protein in contact with Rrp45, Rrp41 and Rrp42 (F). Dis3 and (Csl4, Rrp40, Rrp4) are placed on opposite faces of the ring to construct the 3D model. 3D, three dimensional; MS, mass spectrometry.