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Genetic evidence for the importance of protein acetylation and protein deacetylation in the halophilic archaeon Haloferax volcanii.

ABSTRACT: Protein acetylation and deacetylation reactions are involved in many regulatory processes in eukaryotes. Recently, it was found that similar processes occur in bacteria and archaea. Sequence analysis of the genome of the haloarchaeon Haloferax volcanii led to the identification of three putative protein acetyltransferases belonging to the Gcn5 family, Pat1, Pat2, and Elp3, and two deacetylases, Sir2 and HdaI. Intriguingly, the gene that encodes HdaI shares an operon with an archaeal histone homolog. We performed gene knockouts to determine whether the genes encoding these putative acetyltransferases and deacetylases are essential. A sir2 deletion mutant was able to grow normally, whereas an hdaI deletion mutant was nonviable. The latter is consistent with the finding that trichostatin A, a specific inhibitor of HdaI, inhibits cell growth in a concentration-dependent manner. We also showed that each of the acetyltransferases by itself is dispensable for growth but that deletion of both pat2 and elp3 could not be achieved. The corresponding genes are therefore "synthetic lethals," and the protein acetyltransferases probably have a common and essential substrate.

SUBMITTER: Altman-Price N

PROVIDER: S-EPMC2648188 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Genetic evidence for the importance of protein acetylation and protein deacetylation in the halophilic archaeon Haloferax volcanii.

Altman-Price Neta N Mevarech Moshe M

Journal of bacteriology 20081229 5

Protein acetylation and deacetylation reactions are involved in many regulatory processes in eukaryotes. Recently, it was found that similar processes occur in bacteria and archaea. Sequence analysis of the genome of the haloarchaeon Haloferax volcanii led to the identification of three putative protein acetyltransferases belonging to the Gcn5 family, Pat1, Pat2, and Elp3, and two deacetylases, Sir2 and HdaI. Intriguingly, the gene that encodes HdaI shares an operon with an archaeal histone homo ...[more]

PMID: 19114494

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Project description:A 20S proteasome, composed of alpha(1) and beta subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa alpha(1)beta-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75 degrees C. The alpha(1)beta-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the alpha(1)beta-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha(1)beta-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the beta-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (K(i) = 0.2 and 8 microM, respectively). In addition to the genes encoding the alpha(1) and beta subunits, a gene encoding a second alpha-type proteasome protein (alpha(2)) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the alpha(1)beta-proteasome resulted in the copurification of the alpha(2) protein with the alpha(1) and beta subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha(1)beta-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different alpha subunits in this halophilic archaeon.

Dataset Information

Genetic evidence for the importance of protein acetylation and protein deacetylation in the halophilic archaeon Haloferax volcanii.

Publications

Genetic evidence for the importance of protein acetylation and protein deacetylation in the halophilic archaeon Haloferax volcanii.

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