Dependence of ORC silencing function on NatA-mediated Nalpha acetylation in Saccharomyces cerevisiae

Abstract

N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was N(alpha) acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.

FIG. 1.

NatA activity was required for HM, telomeric, and rDNA silencing and for the integrity of telomeric foci. (A) The deletion of NAT1 resulted in derepression of HML and HMR SS ΔI, as measured by the reduced mating ability of MATa and MATα strains. Patch-mating assays were performed with MATa strains AEY2 (WT) and AEY80 (nat1Δ) and MATα HMR SS ΔI strains AEY5 (WT) and AEY1273 (nat1Δ). (B) Silencing of URA3 inserted near the left telomere of chromosome VII depended on functional NatA. Serial dilutions of strains AEY1017 (WT) and AEY2371 (nat1Δ) were assayed on 5-FOA medium counterselecting for URA3 expressing cells. (C) The association of GFP-tagged Sir3p with telomeric foci was inhibited in nat1Δ cells. Strains AEY160 (WT) and AEY2786 (nat1Δ) were transformed with pAE580. Bar, 2 μm. (D) Silencing of MET15 inserted into the rDNA locus was impaired by nat1Δ, as indicated by the brighter colony color of strain AEY 2786 (nat1Δ) compared to AEY160 (WT) on lead indicator medium.

FIG. 2.

The silencing function of NatA was genetically linked to ORC1. (A) Tethered silencing by Orc1p, but not the other ORC subunits, was independent of NAT1 and required SIR1. In MATα strains AEY1275 (WT), AEY1276 (nat1Δ), and AEY 2947 (nat1Δ sir1Δ), the ORC binding site of the synthetic HMR-E silencer was replaced by five Gal4-binding sites (HMR SS ΔI and 5xGal4-RAP-ABF). The strains carried plasmids encoding the Gal4 DNA-binding domain fused N terminally to Orc1p (5 to 267 amino acids) (pAE408), Orc2p (pAE108), Orc3p (pAE595), Orc4p (pAE597), Orc5p (pAE109), Orc6p (pAE516), and Sir1p (pAE100) and were tested for HMR silencing in patch-mating assays. (B) The deletion of the binding site for Rap1p, but not for ORC or Abf1p, from HMR-E disrupted HMR silencing in nat1Δ mutants. HMR silencing was tested by the α-mating ability of wild-type and nat1Δ strains with wild-type HMR-E (AEY1 and AEY1227) and HMR-E lacking the binding site for ORC (AEY84 and AEY2146), Rap1p (AEY81 and AEY2144), and Abf1p (AEY71 and AEY2148). The results from quantitative mating assays are given relative to a value of 1.0 for AEY1 (WT).

FIG. 3.

Orc1p was N terminally acetylated by NatA. (A) The isoelectric point (pI) of the Orc1p N terminus shifted to a more basic pH either by the deletion of NAT1 or by the mutation of the penultimate alanine to valine or proline. Whole-cell protein extracts of strains AEY2719 (WT), AEY2758 (nat1Δ), AEY3107 (orc1-A2P), and AEY3110 (orc1-A2V) were applied to IEF and SDS gels. TAP-tagged Orc1p (amino acids 1 to 250) was detected in subsequent immunoblots with a PAP-anitbody. The faster-migrating band in the SDS gel was identified as Orc1p by MALDI-TOF analysis and probably is a proteolytic fragment. (B) Theoretical molecular mass of N-terminal peptides of Orc1p generated by proteolysis with AspN or GluC endopeptidase. The molecular mass as calculated by using http://us.expasy.org/tools/peptide-mass.html increases by 42 Da due to N^α acetylation. (C) MALDI-TOF spectra of Orc1-TAP derived from a wild-type, but not from a nat1Δ strain, identified the mass of an acetylated N-terminal peptide of Orc1p. Orc1-TAP was purified for MALDI-TOF analysis from AEY2719 (WT) and AEY2758 (nat1Δ). The data obtainedfrom the AspN and GluC cleaved samples were consistent for each strain with minimal differences to the theoretical value due to the precision of measurements. (D) The MALDI-TOF spectrum of Orc1-TAP from the nat1Δ strain, but not from wild-type strain, contained the mass of an unacetylated N-terminal Orc1 peptide. Analysis was performed as in Fig. 3C. (E) Orc1p was not destabilized by nat1Δ. Whole-cell protein extracts of wild-type (NAT1) (AEY3068) and nat1Δ (AEY 3070) strains expressing HA-tagged Orc1p were analyzed by Western blotting with an α-HA antibody. Equal protein concentrations were loaded on lanes 1 and 4, 2 and 5, and 3 and 6, respectively.

FIG. 4.

N^α acetylation of Orc1p was essential for telomeric silencing. (A) A URA3 gene inserted near the left telomere of chromosome VII was derepressed in unacetylated orc1-A2P and orc1-A2V mutants. URA3 expression was tested in serial dilution assays of strains AEY1017 (ORC1), AEY3038 (orc1-A2V), AEY3105 (orc1-A2P), and AEY2371 (nat1Δ) on 5-FOA-containing medium. (B) Telomeric association of Orc1 was not affected by nat1Δ. Anti-HA ChIPs from ORC1-HA strains AEY3068 (NAT1) and AEY3070 (nat1Δ) were analyzed by PCR with primers specific to the telomeric CoreX element of chromosome XI-L (TEL) and as a negative control to the SSC1 gene. The inverse image of an ethidium bromide-stained gel shows the products of 1:100-diluted input samples (I) and of 1:4 diluted samples that were either precipitated with α-HA antibody (+) or mock treated (−). (C) Loss of N^α acetylation of Orc1p did not impair silencing of HML and HMR SSΔI. Patch-mating assays were performed to test HML silencing by using MATa strains AEY2867 (ORC1), AEY3102 (orc1-A2P), AEY2913 (orc1-A2V), and AEY2912 (nat1Δ) and to test HMR SS ΔI silencing by using the MATα strains AEY2866 (ORC1), AEY3103 (orc1-A2P), AEY2903 (orc1-A2V), and AEY2916 (nat1Δ). (D) nat1Δ, but not unacetylated orc1 mutants caused slight derepression of ADE2 inserted at the HMR locus. Serial dilutions of strains AEY743 (WT), AEY3101 (orc1-A2P), AEY2721 (orc1-A2V), and AEY3109 (nat1Δ) were grown on medium lacking adenine.

FIG. 5.

nat1Δ was synthetically lethal with orc2-1 and SUM1-1. (A) Unacetylated orc1-A2V and orc1-A2P mutants were not temperature sensitive. Serial dilutions of strains AEY2866 (ORC1), AEY 3103 (orc1-A2P), AEY2903 (orc1-A2V), and AEY2916 (nat1Δ) were grown for 2 days on complete medium at the indicated temperatures. (B) Viability of the orc2-1 nat1Δ double mutants was rescued by plasmid-borne ORC2. AEY3161 (orc2-1 nat1Δ pURA3-ORC2) transformed either with pJR1818 (pHIS3-ORC2) (12) or with pRS313 (vector) was tested for ORC2 dependence by counter selection for pURA3-ORC2 on 5-FOA medium. (C) SUM1-1 nat1Δ double mutants were inviable. SUM1-1 nat1Δ segregants of tetrads dissected from a cross between SUM1-1 (AEY1224) and nat1Δ (AEY3008) are marked by arrows.

FIG. 6.

Sir3p was N terminally acetylated by NatA. (A) The isoelectric point of the Sir3p N terminus was shifted toward a more basic pH upon the deletion of NAT1 and when the penultimate amino acid of Sir3 was changed to proline but not to threonine. Whole-cell extracts of strains expressing TAP-tagged N-terminal peptides (amino acids 1 to 235) of Sir3p (AEY3171 [WT]) and AEY3173 [nat1Δ]), Sir3-A2T (AEY3334), and Sir3-A2P (AEY3371) were applied to IEF and SDS gels, which were subsequently analyzed in immunoblots with the PAP antibody. The double band of the Sir3-A2P sample in the IEF gel suggested the coexistence of N^α-acetylated and unacetylated protein. (B) The sir3-A2P mutation caused derepression at HML and HMR controlled by the synthetic HMR-E silencer (HMR SS ΔI), as measured in patch mating assays of MATa strains AEY2867 (SIR3) and AEY3373 (sir3-A2P) and MATα strains AEY2866 (WT) and AEY3378 (sir3-A2P), respectively.