Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast.

Abstract

A gene from Saccharomyces cerevisiae has been mapped, cloned, sequenced and shown to encode a catalytic subunit of an N‐terminal acetyltransferase. Regions of this gene, NAT1, and the chloramphenicol acetyltransferase genes of bacteria have limited but significant homology. A nat1 null mutant is viable but exhibits a variety of phenotypes, including reduced acetyltransferase activity, derepression of a silent mating type locus (HML) and failure to enter G0. All these phenotypes are identical to those of a previously characterized mutant, ard1. NAT1 and ARD1 are distinct genes that encode proteins with no obvious similarity. Concomitant overexpression of both NAT1 and ARD1 in yeast causes a 20‐fold increase in acetyltransferase activity in vitro, whereas overexpression of either NAT1 or ARD1 alone does not raise activity over basal levels. A functional iso‐1‐cytochrome c protein, which is N‐terminally acetylated in a NAT1 strain, is not acetylated in an isogenic nat1 mutant. At least 20 other yeast proteins, including histone H2B, are not N‐terminally acetylated in either nat1 or ard1 mutants. These results suggest that NAT1 and ARD1 proteins function together to catalyze the N‐terminal acetylation of a subset of yeast proteins.

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…In contrast, deletion of NAT1 in a [PSI ϩ ] strain reversed the prion phenotype, leading to the formation of pink colonies on rich media and the inability to grow in the absence of adenine ( Figure 1C). Double mutant ⌬ard1⌬nat1 [PSI ϩ ] and [psi Ϫ ] strains were phenotypically indistinguishable from the single disruptions ( Figure 1C), consistent with the roles of Ard1 and Nat1 as subunits of a single enzyme complex (Mullen et al, 1989;Park and Szostak, 1992).…”

“…In contrast, deletion of NAT1 in a [PSI ϩ ] strain reversed the prion phenotype, leading to the formation of pink colonies on rich media and the inability to grow in the absence of adenine ( Figure 1C). Double mutant ⌬ard1⌬nat1 [PSI ϩ ] and [psi Ϫ ] strains were phenotypically indistinguishable from the single disruptions ( Figure 1C), consistent with the roles of Ard1 and Nat1 as subunits of a single enzyme complex (Mullen et al, 1989;Park and Szostak, 1992).Overproduction of Nat1 has previously been linked to chromosome instability (Ouspenski et al, 1999), raising the possibility that another unknown genetic lesion leads to the observed reversal of the prion phenotype in NatA null strains. However, the NAT1 or ARD1 disruptions cosegregated with adenine auxotrophy in 100% of tetrads analyzed (11 or 16 tetrads, respectively), indicating tight linkage.…”

“…Thus, the ARD1 gene alters the expressivity of the ade1-14 allele in a [PSI ϩ ]-dependent manner. The NatA acetyltransferase is a heterotrimer composed of Ard1, the catalytic subunit (Polevoda et al, 1999), Nat1, a factor essential for NatA activity that tethers the complex to the ribosome for cotranslational modification of substrates (Mullen et al, 1989;Park and Szostak, 1992;Gautschi et al, 2003), and Nat5, a subunit dispensable for NatA function (Gautschi et al, 2003). To further explore the importance of NatA activity on the [PSI ϩ ] phenotype, we disrupted NAT1 and NAT5 alone or in combination with ARD1 disruptions.…”

Nα‐acetyltransferase deficiency alters protein synthesis in Saccharomyces cerevisiae

“…We propose that the synthesis of these proteins may result from derepression or activation of genes regulated by regulatory proteins, which are no longer acetylated, as has been suggested to be the case for a protein regulating aspecific mating type genes in the aaal mutant [17,19].…”

“…The enzyme is encoded by a single gene (AAA I; also called NA 77 [ 171) [ 181. A null mutation deficiency created by gene replace-ment (au&), while not lethal, makes cells grow slowly and heterogeneously, sporulate defectively, become sensitive to heat shock, fail to enter the stationary phase, and display reduced a-type functions [17,19]. In order to study the effect of N"-acetyltransferase deficiency on protein synthesis in yeast, a comparison between the soluble proteins, isolated and then separated by two-dimensional gel electrophoresis, from wild type and aaal mutant was carried out by computer-based analysis of two-dimensional protein gels.…”