The cDNAs encoding two major subunits, named YC1 and YC7-alpha, of yeast proteasomes (multicatalytic proteinase complexes) were isolated and sequenced. As deduced from their nucleotide sequences, YC1 and YC7-alpha consist of 288 and 252 amino acid residues with calculated molecular weights of 31,534 and 27,999, respectively. They showed marked sequence homology to other eukaryotic proteasome components, suggesting that proteasomes are composed of a family of subunits with the same evolutional origin. To obtain information on the physiological role of proteasomes, we disrupted the chromosomal genes of YC1 and YC7-alpha of yeast cells independently, using isolated cDNA clones. Disruption of the coding region of one copy of the YC1 gene in diploid yeast created a recessive lethal mutation, but disruption of the 3'-noncoding region of the gene had no effect on cell proliferation. Disruption of the YC7-alpha gene also had a lethal effect on haploid yeast cells. These findings demonstrated that YC1 and YC7-alpha are both encoded by a single copy gene and that these genes are essential for proliferation of yeast cells.