A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.
A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.
Donovan
M G
MG
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Veldman
S A
SA
Bodley
J W
JW
D003101:Collodion; D004167:Diphtheria Toxin / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D005656:Fungal Proteins / Q000187:drug effects / Q000378:metabolism*; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis; D016296:Mutagenesis; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
D004167:Diphtheria Toxin; D005656:Fungal Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006639:Histidine; C027527:diphthamide; D003101:Collodion; D011065:Poly(ADP-ribose) Polymerases