Volume 2, Issue 5, Pages 639-651 (November 1998) Two Actin-Related Proteins Are Shared Functional Components of the Chromatin- Remodeling Complexes RSC and SWI/SNF  Bradley R Cairns, Hediye Erdjument-Bromage, Paul Tempst, Fred Winston, Roger D Kornberg  Molecular Cell  Volume 2, Issue 5, Pages 639-651 (November 1998) DOI: 10.1016/S1097-2765(00)80162-8

Figure 1 SWI/SNF and RSC Complexes Contain Two Proteins Similar to Actin (A) Purified SWI/SNF and RSC. For SWI/SNF, the immune eluate from an anti-Snf6 immuno-affinity column (described in Cairns et al. 1994) is shown stained with silver. For RSC, Fraction #36 from from Mono S (described in Cairns et al. 1996c) is shown stained with Coomassie. (B) Actin is divided into four structural domains. The locations of ATP and the divalent ion (large bullet) are indicated. Adapted from Kabsch et al. 1990 with permission. (C) Alignments of actin (S. cerevisiae Act1) with Arp7, Arp9, and human Baf53 reveal similarity of the yeast Arp proteins to actin in domains 1 and 3, but not 2 or 4. At left is a diagram showing the order of the domains shown in (B) as the actin main chain is traced through the alignments. The locations of the mutations conferring temperature sensitivity (isolated by random mutagenesis) are shown as bullets (·), and the locations chosen for site-directed replacements with asterisks (*). Positions showing amino acid similarity are shaded Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 2 Arp7 and Arp9 Cofractionate with RSC or SWI/SNF For both SWI/SNF and RSC purification, yeast extracts were initially fractionated on Bio-Rex 70, DEAE Sephacel, hydroxylapatite, and Mono Q (Cairns et al. 1994, Cairns et al. 1996c) prior to resolution on the columns indicated. (A) Arp7 and Arp9 cofractionate on Mono S and TSK-heparin, and coelute with the RSC. For TSK-heparin (upper panel), adsorbed proteins were eluted in a buffer with a linear gradient of 200–800 mM potassium acetate. For Mono S (lower panel), adsorbed proteins were eluted in a buffer with a linear gradient of 100–800 mM potassium acetate. For each, fractions (approximately 2.5 μg per lane) were separated in an SDS-10% acrylamide gel and immunoblotted with anti-Rsc6p, anti-Arp7, and anti-Arp9 antiserum. (B) Arp7 and Arp9 cofractionate on TSK-heparin with SWI/SNF complex. Adsorbed proteins were eluted in a buffer with a linear gradient of 200–800 mM potassium acetate. For each, fractions (approximately 2.0 μg per lane) were separated in an SDS-10% acrylamide gels and immunoblotted separately with anti-Snf6, anti-Arp7, or anti-Arp9 antiserum. Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 3 Arp7 Antibodies Immunodeplete Arp9 Protein and Other Members of RSC (A) Arp7 and Arp9 antibodies are specific for their antigen. Pure RSC (Mono S fraction #36, 250 ng) was separated on two neighboring lanes of a SDS-10% acrylamide gel and immunoblotted with anti-Rsc6 antiserum and either anti-Arp7 antiserum (lane 1) or anti-Arp9 antiserum (lane 2). (B) Immunoprecipitation of RSC with anti-Arp7 antibodies. A fraction containing highly purified RSC (heparin fraction #38, 1 μg) was treated with either Anti-Arp7 antibodies or anti-Snf6 antibodies conjugated to protein A–Sepharose. The following were separated in a SDS-10% acrylamide gel and immunoblotted: pure RSC (Mono S fraction #36, 250 ng, lane 1), heparin RSC (Fraction #38, 500 ng, lane 2), half of the anti-Arp7 supernatant (lane 3), half of the anti-Arp7 immune eluate (lane 4), half of the anti-Snf6 supernatant (lane 5), or half of the anti-Snf6 immune eluate (lane 6). (C) Immunoprecipitation of RSC with anti-Arp9 antibodies. RSC (heparin fraction #38, 1 μg) was treated with either Anti-Arp9 antibodies or anti-Snf6 antibodies conjugated to protein A–Sepharose. The following were separated in a SDS-10% acrylamide gel and immunoblotted: pure RSC (Mono S fraction #36, 250 ng, lane 1), heparin RSC (500 ng, lane 2), half of the anti-Arp9 supernatant (lane 3), half of the anti-Arp9 immune eluate (lane 4), half of the anti-Snf6 supernatant (lane 5), or half of the anti-Snf6 immune eluate (lane 6). Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 4 ARP7 and ARP9 Are Essential for Mitotic Growth in S288C Genetic Background and Important for Growth in W303 Genetic Background Diploids heterozygous for either an arp7Δ mutation or an arp9Δ mutation were sporulated and dissected. Four representative tetrads are shown, with the four spores (A–D) from each tetrad in a horizontal row. (A) ARP7 and ARP9 are essential in S288C background. Dissections of the S288C heterozygous diploids YBC16 (arp7Δ::LEU2/ARP7, top panel) and YBC20 (arp9Δ::LEU2/ARP9, lower panel). More extensive growth of S288C tetrads (ten days) did not reveal microcolonies with an arpΔ genotype. (B) ARP7 and ARP9 are not essential for growth in W303 background. Dissections of W303 heterozygous diploids BCY340 (arp7Δ::LEU2/ARP7, top panel) and BCY330 (arp9Δ::LEU2/ARP9, lower panel). Tetrads were grown for eight days at 30°C. Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 5 Temperature-Sensitive Mutations in ARP7 and ARP9 (A) Growth of arp7 Ts− strains at permissive (30°C) and nonpermissive (37°C) temperatures. Strains: WT, BY4727; or YBC726 transformants harboring 2 μm ARP7 (pY24LIB.ARP7), ARP7 (pNCT.ARP7) (wild type); those bearing arp7 Ts− mutations contain pNCT.arp7–122 (YBC776), pNCT.arp7–161 (YBC777), and pNCT.-arp7–162 (YBC778). (B) Growth of the Ts− arp9–661 strain at permissive (30°C) and nonpermissive (37°C) temperature. Strains: WT, BY4727; arp9–661, YBC775 harboring pNCT.arp9–661; arp9Δ, YBC790 harboring pNCT.ARP9. All strains are S288C derivatives. Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 6 Ts− Mutations in ARP7 and ARP9 Confer a Moderate Spt− Phenotype (A) Growth of strains bearing Ts− arp7 mutations and the δ element insertion alleles his4–912δ and lys2–128δ. Strains were grown as patches on YPD, replica-plated to synthetic media lacking either histidine or lysine and grown for five days at 30°C. Strains: all are YBC726 harbouring plasmids with the indicated arp7 Ts− mutations except wild type (WT, FY120) and spt6–14 (FY957). (B) Growth of the Ts− arp9–661 strain. YBC780 harboring the plasmid pNCT.arp6–661 or pNCT. ARP9. The wild-type (WT) strain is FY2. All strains are S288C derivatives. Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)

Figure 7 ARP7 Mutations and Their Phenotypes Superimposed on the Crystal Structure of Actin The locations of the alterations in Arp7 and Arp9 are based on the sequence alignments presented in Figure 1C. Red dots identify the locations of the arp7 Ts− substitutions. Green dots identify the locations of site-directed replacements performed with both ARP7 and ARP9 that were designed to impair ATP hydrolysis (all of which conferred no phenotype). The large blue dot identifies the location of the divalent cation. Adapted from Kabsch et al. 1990 with permission. Molecular Cell 1998 2, 639-651DOI: (10.1016/S1097-2765(00)80162-8)