A combinatorial depletion strategy is combined with biochemistry, quantitative proteomics and computational approaches to elucidate the structure of the SAGA/ADA complexes. The analysis reveals five connected functional modules capable of independent assembly.
A combinatorial approach of gene depletions with multiple bait proteins coupled with biochemical, proteomic and computational approaches can experimentally determine modules of stable multi-protein complexes.SAGA is a 19-subunit complex consisting of five connected modules with Spt20 being particularly important for the assembly of the intact complex.One of the modules, the HAT/Core module, is also shared with the distinct six-subunit complex ADA.Architectural models of large multi-protein complexes can be assembled using our approach, which is an alternative method to generate novel insight into the organization and architecture of multi-protein complexes.
Determining the architectures of protein complexes improves our understanding of protein cellular functions. In order to efficiently characterize the subunits of protein complexes assembled in vivo, affinity purification followed by proteomics mass spectrometry (APMS) strategies have been devised. Partial or whole protein complexes are first biochemically isolated using tagged components of the complex, followed by an identification of all co-purified proteins using mass spectrometry. However, those approaches are insufficient to provide information about the spatial arrangement and the interrelationship of the proteins of the respective complex.
In this study, we developed and applied a novel method utilizing biochemistry, quantitative proteomics and computational approaches in order to characterize the organization of proteins in a complex. The key of our method is the systematic purification of several tagged components of the protein complex in multiple genetic deletion strains, which serve to compromise the integrity of the complex. Using a series of computational methods, these raw quantitative values are next interpreted in order to determine the modular organization of the complex as well as the interrelationships between its subunits, which in turn can be used to predict a macromolecular model of the complex.
We tested this approach to obtain novel insights into the architecture of multi-protein complexes on the Saccharomyces cerevisiae Spt–Ada–Gcn5 histone acetyltransferase (HAT) (SAGA) and ADA complexes, which are conserved complexes involved in chromatin remodeling (Koutelou et al, 2010). Regular quantitative APMS strategies in wild-type backgrounds were not sufficient to separate tight protein complexes like SAGA/ADA into its distinct modules. However, after perturbing the system using genetic deletions of several subunits located in different topological parts of SAGA, hierarchical cluster analysis performed on 34 purifications (generated using 10 different TAP-tagged baits) resulted in a dissociation of the Gcn5 HAT complexes into five modules: (1) the SA_TAF module, (2) the SA_SPT module, (3) the DUB module, (4) the HAT/Core module and (5) the ADA module (Figure 2A and B).
The approach of purifying a protein in a deletion strain furthermore provides valuable information about the influence of the deleted subunit on the association and interdependency of the bait and the remaining preys. In order to quantify these associations, we calculated a probability between every prey and bait in the deletion strain purifications based on Bayes' theorem (Sardiu et al, 2008). In conjunction with preexisting interaction data obtained from yeast two-hybrid and genetic complementation assays, we finally used these probabilities to predict a low-resolution model for the architecture of the SAGA and ADA complexes (Figure 4).
This novel approach revealed that the SAGA/ADA complexes are composed of five distinct functional modules, of which two were not previously described (SA_SPT and SA_TAF). These modules, which are responsible for different functions of the SAGA complex, are capable of assembling independently from the remaining modules of the complex. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 HAT complexes. Compared with other structural studies, which mapped 9 of the 19 known SAGA subunits using single EM reconstruction (Wu et al, 2004) or resolved the structure of the 4 subunits of the DUB module using X-ray crystallography (Kohler et al, 2010; Samara et al, 2010), our approach is not limited to a maximum number of complex subunits. Consequently, we were able to construct a macromolecular model consisting of all 21 SAGA/ADA subunits, which bridges the gap between the previous limited EM analysis and focused X-ray crystallography analysis.
Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt–Ada–Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.