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Panorama of ancient metazoan macromolecular complexes

View Article: PubMed Central - PubMed

ABSTRACT

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we then generated a draft conservation map consisting of >1 million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering revealed a spectrum of conservation, ranging from ancient Eukaryal assemblies likely serving cellular housekeeping roles for at least 1 billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, by affinity-purification and by functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic significance and adaptive value to animal cell systems.

No MeSH data available.


Additional validation dataa, Confirmation of MIB2 interactions by co-immunoprecipitation. Extract (~10 mg protein) from cultured human HCT116 cells expressing FLAG-tagged MIB2 or control (WT) cells was incubated with 100 μl anti-FLAG M2 resin for 4 h by gently rotating at 4°C. After extensive washing with RIPA buffer, co-purifying proteins bound to the beads were eluted by the addition of 25 μl Laemmli loading buffer at 95 °C. Polypeptides were separated by SDS-PAGE and immunoblotted using FLAG, VPS4A, VPS4B or IST1 antibodies as indicated (expanded gel images provided in SI). b, Protein co-complex interactions reported in the CYC2008 yeast protein complex database42 are reconstructed accurately from the co-fractionation data, regardless of whether the full set of co-fractionation plus external data are used to derive protein interactions (‘All data’, see also Fig. 4b) or if the external yeast data was specifically excluded from the analyses (‘All data, excluding yeast’).
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Figure 11: Additional validation dataa, Confirmation of MIB2 interactions by co-immunoprecipitation. Extract (~10 mg protein) from cultured human HCT116 cells expressing FLAG-tagged MIB2 or control (WT) cells was incubated with 100 μl anti-FLAG M2 resin for 4 h by gently rotating at 4°C. After extensive washing with RIPA buffer, co-purifying proteins bound to the beads were eluted by the addition of 25 μl Laemmli loading buffer at 95 °C. Polypeptides were separated by SDS-PAGE and immunoblotted using FLAG, VPS4A, VPS4B or IST1 antibodies as indicated (expanded gel images provided in SI). b, Protein co-complex interactions reported in the CYC2008 yeast protein complex database42 are reconstructed accurately from the co-fractionation data, regardless of whether the full set of co-fractionation plus external data are used to derive protein interactions (‘All data’, see also Fig. 4b) or if the external yeast data was specifically excluded from the analyses (‘All data, excluding yeast’).

Mentions: We used multiple approaches to assess the accuracy (Fig. 4) and functional significance (Fig. 5) of the predicted complexes. First, we performed affinity purification-mass spectrometry (AP/MS) experiments on select novel complexes from the ‘new’, ‘old’ and ‘mixed’ age clusters, validating most associations in both worm and human (Fig. 4a, Extended Data Fig. 6a). We next performed a global validation by comparing our derived complexes to a newly reported large-scale AP/MS study of 23,756 putative human protein interactions detected in cell culture (BioGrid pre-publication 166968, Huttlin EL et al., downloaded Feb. 10, 2015), and observed a partial, but exceptionally significant, overlap to a degree comparable to literature-derived complexes (Fig. 4b, Extended Data Fig. 6b).


Panorama of ancient metazoan macromolecular complexes
Additional validation dataa, Confirmation of MIB2 interactions by co-immunoprecipitation. Extract (~10 mg protein) from cultured human HCT116 cells expressing FLAG-tagged MIB2 or control (WT) cells was incubated with 100 μl anti-FLAG M2 resin for 4 h by gently rotating at 4°C. After extensive washing with RIPA buffer, co-purifying proteins bound to the beads were eluted by the addition of 25 μl Laemmli loading buffer at 95 °C. Polypeptides were separated by SDS-PAGE and immunoblotted using FLAG, VPS4A, VPS4B or IST1 antibodies as indicated (expanded gel images provided in SI). b, Protein co-complex interactions reported in the CYC2008 yeast protein complex database42 are reconstructed accurately from the co-fractionation data, regardless of whether the full set of co-fractionation plus external data are used to derive protein interactions (‘All data’, see also Fig. 4b) or if the external yeast data was specifically excluded from the analyses (‘All data, excluding yeast’).
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Figure 11: Additional validation dataa, Confirmation of MIB2 interactions by co-immunoprecipitation. Extract (~10 mg protein) from cultured human HCT116 cells expressing FLAG-tagged MIB2 or control (WT) cells was incubated with 100 μl anti-FLAG M2 resin for 4 h by gently rotating at 4°C. After extensive washing with RIPA buffer, co-purifying proteins bound to the beads were eluted by the addition of 25 μl Laemmli loading buffer at 95 °C. Polypeptides were separated by SDS-PAGE and immunoblotted using FLAG, VPS4A, VPS4B or IST1 antibodies as indicated (expanded gel images provided in SI). b, Protein co-complex interactions reported in the CYC2008 yeast protein complex database42 are reconstructed accurately from the co-fractionation data, regardless of whether the full set of co-fractionation plus external data are used to derive protein interactions (‘All data’, see also Fig. 4b) or if the external yeast data was specifically excluded from the analyses (‘All data, excluding yeast’).
Mentions: We used multiple approaches to assess the accuracy (Fig. 4) and functional significance (Fig. 5) of the predicted complexes. First, we performed affinity purification-mass spectrometry (AP/MS) experiments on select novel complexes from the ‘new’, ‘old’ and ‘mixed’ age clusters, validating most associations in both worm and human (Fig. 4a, Extended Data Fig. 6a). We next performed a global validation by comparing our derived complexes to a newly reported large-scale AP/MS study of 23,756 putative human protein interactions detected in cell culture (BioGrid pre-publication 166968, Huttlin EL et al., downloaded Feb. 10, 2015), and observed a partial, but exceptionally significant, overlap to a degree comparable to literature-derived complexes (Fig. 4b, Extended Data Fig. 6b).

View Article: PubMed Central - PubMed

ABSTRACT

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we then generated a draft conservation map consisting of >1 million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering revealed a spectrum of conservation, ranging from ancient Eukaryal assemblies likely serving cellular housekeeping roles for at least 1 billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, by affinity-purification and by functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic significance and adaptive value to animal cell systems.

No MeSH data available.