Isolation of a Gene Encoding a Chaperonin-Like Protein by Complementation of Yeast Amino Acid Transport Mutants with Human cDNA

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 13; pp. 6060 - 6064
• Main Authors: Segel, George B., Boal, Thomas R., Cardillo, Thomas S., Murant, Francis G., Lichtman, Marshall A., Sherman, Fred
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.07.1992; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; Amino acids; Ammonia - pharmacology; Base Sequence; Biochemistry; Biological Transport; Blotting, Southern; cDNA; Chaperonins; Citrulline - metabolism; Cloning, Molecular; Complementary DNA; Complementation; DNA; DNA - genetics; Genes; Genetic Complementation Test; Genetics; Genomics; histidine; Histidine - metabolism; Humans; like; man; Medical research; Molecular Sequence Data; mutants; Plasmids; predictions; Proteins; Proteins - genetics; Quaternary ammonium compounds; Repression; Restriction Mapping; RNA, Messenger - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Alignment; transformation; transport; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.13.6060

A human cDNA library in λ-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine per-mease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1-strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.