FIGURE 1.

Subunit-interacting network analysis suggests two copies of the essential Rco1 subunit within Rpd3S. A–D, Coomassie staining of the indicated recombinant Rpd3S complexes that were produced in a baculovirus expression system. The bands corresponding to each subunit were indicated either as green dots (the tagged subunits for purification) or red dots (untagged subunits). Indicated Rpd3S complexes were prepared via FLAG purification. − indicates that the particular virus was omitted from the reconstitution. Asterisks indicate degradation or contaminated proteins. E, Eaf3 is in a monomeric form in Rpd3S. The indicated viruses were co-infected to insect cells. Western blots were performed after FLAG purification. F, Rpd3S contains two copies of Rco1. Recombinant Rpd3S was prepared through FLAG and HA tandem purification and stained with Coomassie Blue. G, a multiple sequence alignment of Rco1 orthologs. The following sequences were used: SpCph2 (Q09698, Schizosaccharomyces pombe), SpCph1(Q09819, S. pombe), SjCph2(SJAG_01110, Schizosaccharomyces japonicas), SjCph1(SJAG_03884, S. japonicas), SkuRco1 (protSku1303, Saccharomyces kudriavzevii), SbaRco1 (Sbay_66.15, Saccharomyces bayanus), ScRco1 (Q04779, Saccharomyces cerevisiae), and SpaRco1 (protSpa836, Saccharomyces paradoxus). Conserved residues are colored with the ClustalX scheme in Jalview (56). The resulting graph was further condensed and is displayed. Conservation value and phylogenetic tree were also calculated in Jalview. The domain structure of Rco1 is illustrated at the bottom, and the critical boundary residues were labeled, although the sequence gaps within Rco1 were not removed.