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.

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We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hsIVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome (3 subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coi expressing heat-shock Proteasomes are multicatalytic proteolytic complexes present in both the nucleus and cytosol of eukaryotic cells (1). The 26S form of the proteasome catalyzes the degradation of ubiquitinconjugated proteins (2-5), and thus it plays a key role in many cellular processes, including progression through the cell cycle (6, 7), removal of abnormal proteins, and antigen presentation (8). The proteolytic core of the 26S complex is the 20S (700 kDa) proteasome particle, which consists of four sevenmembered rings. The subunits of the 20S proteasome fall into two families (9, 10): the a-type forms the two outer rings, and P-type, which contain the active sites, forms the two inner rings of the complex.Proteasomes were thought to exist exclusively in eukaryotes and certain archaebacteria (11). However, 20S proteasomes were recently discovered in the actinomycete Rhodococcus (12), and in the Escherichia coli genome sequencing project, a novel heat-shock locus (hslVU) was discovered that encodes a 19-kDa protein (HslV) (13), whose sequence is similar to 3-type proteasome subunits. This discovery of proteasome-related genes was surprising, because several groups had failed to observe a structure in E. coli resembling the proteasome or proteins resembling ubiquitin. The hslV gene is cotranscribed with the adjacent hslU gene, which codes for a 50-kDa protein containing one ATP/GTP binding motif (13 For the expression of glutathione S-transferase (GST)-fusion proteins, the hslVand hslU genes were PCR amplified separately using A phage 18-126 DNA bearing the hslVU operon, kindly provided by F. Blattner (University of Wisconsin-Madison), and cloned into the vector pGEX-2T (Pharmacia). Vector pV106 (GST-HslV) and pU206 (GST-HslU) were electroporated into E. coli C600 cells. GST-fusion proteins were purified from strains C106 and C206 using the GST Purification Module (Pharmacia). To obtain antibodies, purified GST-HslV and GST-HslU proteins were injected into rabbits. Polyclonal anti-HslV and antiHslU antibodies were then affinity purified using the GST-fusions as ligands, and depleted of anti-GST antibodies using a GST column.Abbreviations: hsl, heat-shock locus; GST, glutathone S-transferase. tTo whom reprint requests should be addressed.5808

Genomic DNA of the yeast, Saceharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-icytochrome c were generated by transforming a cycl mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cycl mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonudeotides as short as 20 nucleotides and with amounts as low as 100 ,ug. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.Site-directed mutagenesis has been an important approach for altering DNA sequences and especially for producing altered forms of proteins. The most versatile methods for producing highly specific changes use single-stranded Escherichia (6,7). Also, Mandecki (8) used synthetic oligonucleotides at least 20 nucleotides long for site-directed mutagenesis by repairing double-strand breaks in E. coli plasmids. In this paper we describe a convenient, single-step procedure of mutagenesis in vivo by which the yeast Saccharomyces cerevisiae are transformed solely with synthetic oligonucleotides.The yeast strain to be used for this procedure should have a target allele bearing a single-site mutation that reverts at a low frequency. In addition, this method requires selection for the transformed yeast. By using the cycl-31 mutant that lacks iso-1-cytochrome c and a series of oligonucleotides that encompass the lesion, revertants having functional, but altered, iso-1-cytochromes c were recovered; these revertants contained sequences corresponding to those of the synthetic oligonucleotides. This procedure has been used to alter the NH2-terminal region of iso-1-cytochrome c and investigate NH2-terminal processing of proteins in yeast. MATERIALS AND METHODSGenetic Nomenclature and Yeast Strains. The symbols CYCI and CYCJ+ denote, respectively, any functional allele and the wild-type allele encoding iso-1-cytochrome c in the yeast S. cerevisiae. The cycl-31 allele causes a complete deficiency of iso-1-cytochrome c, whereas CYCI-793 and CYCI-795 denote two altered CYCI alleles obtained from the cycl-31 mutation with two different synthetic oligonucleotides. CYC7' denotes the wild-type allele encoding iso-2-cytochrome c, and cyc7-67 denotes a partial deletion of the CYC7 locus that results in the complete deficiency of iso-2-cytochrome c. CYC)+ CYC7' strains can grow on both lactate and glycerol media; cycl -31 CYC7' strains can grow on glycerol medium but not lactate medium, whereas cycl-31 cyc7-67 strains cannot grow on either lactate or glycerol media (9, 10). The main yeast strain used in this study, B-7528, has the genotype MATa cycl-31 cyc7-67 ura3-52 lysS-10 and is referred to as the cycl-31...