Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance.
Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.
Bockhorn
Jessica
J
Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
Balar
Bharvi
B
He
Dongming
D
Seitomer
Eden
E
Copeland
Paul R
PR
Kinzy
Terri Goss
TG
D000483:Alleles; D000596:Amino Acids; D003542:Cystathionine gamma-Lyase / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006238:Haploidy; D056892:Mediator Complex; D050296:Microbial Viability / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012645:Selenomethionine / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics
D000596:Amino Acids; D056892:Mediator Complex; C093967:SSN2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012645:Selenomethionine; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase