The Nuclear Actin-related Protein ofSaccharomyces cerevisiae, Act3p/Arp4, Interacts with Core Histones

Abstract

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

Correlation between chromatin association and transcriptional regulation for the Act3p/Arp4 nuclear actin-related protein of Saccharomyces cerevisiae

“…Yeast strains DY2864 (MATa his4-912δ-ADE2 his4-912δ lys2-128δ can1 trp1 ura3 ACT3), DY4285 (MATa his4-912δ -ADE2 lys2-128δ can1 leu2 trp1 ura3 act3-ts26) and DY4519 (MATa his4-912δ -ADE2 lys2-128δ can1 leu2 trp1 ura3 act3-ts12) were used for analyses of chromatin structure. To isolate new temperature-sensitive alleles of ACT3, a plasmid shuffle was performed as described (16,20). DNA sequencing shows that the act3-ts26 and act3-ts12 alleles each have a single amino acid substitution of G187R and G455S, respectively.…”

“…Mononucleosomes were reconstructed in vitro with purified core histones of chicken (16) and a 180 bp DNA fragment recovered from BssHII-digested pBluescript SK+ (Stratagene) according to Tatchell and van Holde (21). Mobility shift assays were performed by mixing the reconstituted mononucleosome and the purified GST fusion protein in 10 µl of binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 5 mM DTT, 5 mM PMSF, 4 mM MgCl 2 , 5% glycerol) for 15 min at 37°C and then by electrophoresis on 1.5% agarose gels in 1× TAE (8 mM Tris, 1 mM sodium acetate, 0.4 mM EDTA, pH 8.0).…”

“…To investigate the involvement of Act3p/Arp4 in transcriptional regulation, the activity of the his4-912δ promoter was tested in two yeast strains possessing conditional act3/arp4 mutations (ts26 and ts12) (16,17). ts26 and ts12 have single amino acid substitutions of G187R and G455S in the genomic ACT3/ARP4 gene, respectively.…”

“…Insertion II, consisting of 83 amino acids, is relatively abundant in charged amino acids and is predicted to form a loop-like structure protruding from the surface of the molecule. We previously reported that insertion II interacts with each of the core histones in vitro (16). Additionally, Act3p/Arp4 is present in the NuA4 histone acetyltransferase complex and the Ino80 chromatin remodeling complex purified from cell extracts (17,18).…”

INO80 subfamily of chromatin remodeling complexes

“…In yeast, this actin/Arp4 module probably has also evolved into a less conserved Arp7/Arp9 dimer found in the SWI/SNF and RSC chromatin remodeling complexes [29]. It has been shown that Arp4 binds to all four histones in vitro and in vivo [30] and Arp8 binds to H3 and H4 with some preference in vitro [10], suggesting that the actin/Arp modules in chromatin modifying complexes might provide chaperone functions for the histone octamer and mediate the rearrangement of histone-histone, and/or histone-DNA contacts during chromatin remodeling. The defined actin-containing module in the INO80 complex provides a unique opportunity to understand the mechanism of nuclear actin.…”