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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.


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Properties of the Commander complexThe automatically-derived 8 subunit Commander complex (Fig. 3b) was subsequently extended to 13 subunits (COMMD1 to 10, CCDC22, CCDC93, and SH3GLB1) based on combined analysis of AP-MS (Fig. 4a), size exclusion chromatograms43 (Fig. 4d), published pairwise interactions30,47,48, and analysis of elution profiles of the remaining COMM domain containing proteins, as shown here. Example protein elution profiles are plotted for Commander complex subunits observed from: a, HEK293 cell nuclear extract; b, sea urchin embryonic (5 days post-fertilization) extract; and c, fly SL2 cell nuclear extract; each fractionated by heparin affinity chromatography. d, Co-expression of Commander complex subunits during embryonic development of X. tropicalis (plotting mean +/− s.d. of 3 clutches; data from ref. 49). e, mRNA expression patterns of Commander complex subunits in stage 15 X. laevis embryos. Images show coordinated spatial expression in early vertebrate embryogenesis, as measured by in situ hybridization (3 embryos examined). f, Knockdown of Commd2 induced marked head and eye defects in developing X. laevis. (top) Commd2 antisense knockdown significantly decreased eye size, shown for stage 38 tadpoles (from 3 clutches; control n = 47 animals, 1 eye each); phenotypes were consistent between translation blocking (MOatg; n = 60) morpholino reagents, splice site blocking (MOsp; n = 50) morpholinos, and knockdowns of interaction partner Commd3 (see Fig. 5a). ***, p < 0.0001, 2-sided Mann-Whitney test. (bottom) Commd2 knockdown induced altered Pax6 patterning in the embryonic eye (control n = 8 animals, 2 eyes each; MO n = 11). g, Commd2/3 knockdown animals show altered neural patterning. Changes in stage 15 X. laevis embryos, measured by in situ hybridization (assayed in duplicates; 5 embryos per treatment), seen upon knockdown but not on controls: the forebrain marker PAX6 was expanded, while the mid-brain marker EN2 was strongly reduced. Strikingly, while expression of KROX20/EGR1 in rhombomere R3 was shifted posteriorly, expression in R5 was strongly reduced or entirely absent. Panels in Fig. 5b are reproduced from this figure and are directly comparable. h, Confirmation of splice-blocking Commd2 morpholino activity. Images and schematic show the basis and results of RT-PCR and agarose gel electrophoresis obtained with the corresponding X. laevis knockdown tadpoles.
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Figure 14: Properties of the Commander complexThe automatically-derived 8 subunit Commander complex (Fig. 3b) was subsequently extended to 13 subunits (COMMD1 to 10, CCDC22, CCDC93, and SH3GLB1) based on combined analysis of AP-MS (Fig. 4a), size exclusion chromatograms43 (Fig. 4d), published pairwise interactions30,47,48, and analysis of elution profiles of the remaining COMM domain containing proteins, as shown here. Example protein elution profiles are plotted for Commander complex subunits observed from: a, HEK293 cell nuclear extract; b, sea urchin embryonic (5 days post-fertilization) extract; and c, fly SL2 cell nuclear extract; each fractionated by heparin affinity chromatography. d, Co-expression of Commander complex subunits during embryonic development of X. tropicalis (plotting mean +/− s.d. of 3 clutches; data from ref. 49). e, mRNA expression patterns of Commander complex subunits in stage 15 X. laevis embryos. Images show coordinated spatial expression in early vertebrate embryogenesis, as measured by in situ hybridization (3 embryos examined). f, Knockdown of Commd2 induced marked head and eye defects in developing X. laevis. (top) Commd2 antisense knockdown significantly decreased eye size, shown for stage 38 tadpoles (from 3 clutches; control n = 47 animals, 1 eye each); phenotypes were consistent between translation blocking (MOatg; n = 60) morpholino reagents, splice site blocking (MOsp; n = 50) morpholinos, and knockdowns of interaction partner Commd3 (see Fig. 5a). ***, p < 0.0001, 2-sided Mann-Whitney test. (bottom) Commd2 knockdown induced altered Pax6 patterning in the embryonic eye (control n = 8 animals, 2 eyes each; MO n = 11). g, Commd2/3 knockdown animals show altered neural patterning. Changes in stage 15 X. laevis embryos, measured by in situ hybridization (assayed in duplicates; 5 embryos per treatment), seen upon knockdown but not on controls: the forebrain marker PAX6 was expanded, while the mid-brain marker EN2 was strongly reduced. Strikingly, while expression of KROX20/EGR1 in rhombomere R3 was shifted posteriorly, expression in R5 was strongly reduced or entirely absent. Panels in Fig. 5b are reproduced from this figure and are directly comparable. h, Confirmation of splice-blocking Commd2 morpholino activity. Images and schematic show the basis and results of RT-PCR and agarose gel electrophoresis obtained with the corresponding X. laevis knockdown tadpoles.

Mentions: We also observed broad agreement between the derived complexes’ inferred molecular weights (assuming 1:1 stiochiometries) and migration by size exclusion chromatography (Fig. 4c; Extended Data Fig. 7a) and density gradient centrifugation (Extended Data Fig. 7b). A prime example is the coherent profiles of a large (~500 kDa) ‘mixed’ complex with several unannotated components (Fig. 4d; Extended Data Fig. 8), dubbed Commander because most subunits share COMM (copper metabolism MURR1) domains30 implicated in copper toxicosis31, among other roles30,32. Commander contains coiled-coil domain proteins CCDC22 and CCDC93 (Figs. 4a, d) in addition to ten COMM domain proteins, broadly supported by co-fractionation in human, fly and sea urchin (Extended Data Fig. 9a–c and Supporting Web Site).


Panorama of ancient metazoan macromolecular complexes
Properties of the Commander complexThe automatically-derived 8 subunit Commander complex (Fig. 3b) was subsequently extended to 13 subunits (COMMD1 to 10, CCDC22, CCDC93, and SH3GLB1) based on combined analysis of AP-MS (Fig. 4a), size exclusion chromatograms43 (Fig. 4d), published pairwise interactions30,47,48, and analysis of elution profiles of the remaining COMM domain containing proteins, as shown here. Example protein elution profiles are plotted for Commander complex subunits observed from: a, HEK293 cell nuclear extract; b, sea urchin embryonic (5 days post-fertilization) extract; and c, fly SL2 cell nuclear extract; each fractionated by heparin affinity chromatography. d, Co-expression of Commander complex subunits during embryonic development of X. tropicalis (plotting mean +/− s.d. of 3 clutches; data from ref. 49). e, mRNA expression patterns of Commander complex subunits in stage 15 X. laevis embryos. Images show coordinated spatial expression in early vertebrate embryogenesis, as measured by in situ hybridization (3 embryos examined). f, Knockdown of Commd2 induced marked head and eye defects in developing X. laevis. (top) Commd2 antisense knockdown significantly decreased eye size, shown for stage 38 tadpoles (from 3 clutches; control n = 47 animals, 1 eye each); phenotypes were consistent between translation blocking (MOatg; n = 60) morpholino reagents, splice site blocking (MOsp; n = 50) morpholinos, and knockdowns of interaction partner Commd3 (see Fig. 5a). ***, p < 0.0001, 2-sided Mann-Whitney test. (bottom) Commd2 knockdown induced altered Pax6 patterning in the embryonic eye (control n = 8 animals, 2 eyes each; MO n = 11). g, Commd2/3 knockdown animals show altered neural patterning. Changes in stage 15 X. laevis embryos, measured by in situ hybridization (assayed in duplicates; 5 embryos per treatment), seen upon knockdown but not on controls: the forebrain marker PAX6 was expanded, while the mid-brain marker EN2 was strongly reduced. Strikingly, while expression of KROX20/EGR1 in rhombomere R3 was shifted posteriorly, expression in R5 was strongly reduced or entirely absent. Panels in Fig. 5b are reproduced from this figure and are directly comparable. h, Confirmation of splice-blocking Commd2 morpholino activity. Images and schematic show the basis and results of RT-PCR and agarose gel electrophoresis obtained with the corresponding X. laevis knockdown tadpoles.
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Figure 14: Properties of the Commander complexThe automatically-derived 8 subunit Commander complex (Fig. 3b) was subsequently extended to 13 subunits (COMMD1 to 10, CCDC22, CCDC93, and SH3GLB1) based on combined analysis of AP-MS (Fig. 4a), size exclusion chromatograms43 (Fig. 4d), published pairwise interactions30,47,48, and analysis of elution profiles of the remaining COMM domain containing proteins, as shown here. Example protein elution profiles are plotted for Commander complex subunits observed from: a, HEK293 cell nuclear extract; b, sea urchin embryonic (5 days post-fertilization) extract; and c, fly SL2 cell nuclear extract; each fractionated by heparin affinity chromatography. d, Co-expression of Commander complex subunits during embryonic development of X. tropicalis (plotting mean +/− s.d. of 3 clutches; data from ref. 49). e, mRNA expression patterns of Commander complex subunits in stage 15 X. laevis embryos. Images show coordinated spatial expression in early vertebrate embryogenesis, as measured by in situ hybridization (3 embryos examined). f, Knockdown of Commd2 induced marked head and eye defects in developing X. laevis. (top) Commd2 antisense knockdown significantly decreased eye size, shown for stage 38 tadpoles (from 3 clutches; control n = 47 animals, 1 eye each); phenotypes were consistent between translation blocking (MOatg; n = 60) morpholino reagents, splice site blocking (MOsp; n = 50) morpholinos, and knockdowns of interaction partner Commd3 (see Fig. 5a). ***, p < 0.0001, 2-sided Mann-Whitney test. (bottom) Commd2 knockdown induced altered Pax6 patterning in the embryonic eye (control n = 8 animals, 2 eyes each; MO n = 11). g, Commd2/3 knockdown animals show altered neural patterning. Changes in stage 15 X. laevis embryos, measured by in situ hybridization (assayed in duplicates; 5 embryos per treatment), seen upon knockdown but not on controls: the forebrain marker PAX6 was expanded, while the mid-brain marker EN2 was strongly reduced. Strikingly, while expression of KROX20/EGR1 in rhombomere R3 was shifted posteriorly, expression in R5 was strongly reduced or entirely absent. Panels in Fig. 5b are reproduced from this figure and are directly comparable. h, Confirmation of splice-blocking Commd2 morpholino activity. Images and schematic show the basis and results of RT-PCR and agarose gel electrophoresis obtained with the corresponding X. laevis knockdown tadpoles.
Mentions: We also observed broad agreement between the derived complexes’ inferred molecular weights (assuming 1:1 stiochiometries) and migration by size exclusion chromatography (Fig. 4c; Extended Data Fig. 7a) and density gradient centrifugation (Extended Data Fig. 7b). A prime example is the coherent profiles of a large (~500 kDa) ‘mixed’ complex with several unannotated components (Fig. 4d; Extended Data Fig. 8), dubbed Commander because most subunits share COMM (copper metabolism MURR1) domains30 implicated in copper toxicosis31, among other roles30,32. Commander contains coiled-coil domain proteins CCDC22 and CCDC93 (Figs. 4a, d) in addition to ten COMM domain proteins, broadly supported by co-fractionation in human, fly and sea urchin (Extended Data Fig. 9a–c and Supporting Web Site).

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 &gt;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.


Related in: MedlinePlus