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A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae : Discrimination of diphtheria toxin‐resistant mutants
Author(s) -
Donovan Maura G.,
Veldman Sarah A.,
Bodley James W.
Publication year - 1992
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320080806
Subject(s) - diphtheria toxin , biology , spheroplast , saccharomyces cerevisiae , biochemistry , lysis , intracellular , yeast , toxin , mutant , elongation factor , lysozyme , microbiology and biotechnology , gene , escherichia coli , ribosome , rna
A genetal method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast 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 nitrocllulose under non‐denaturing conditions and potentially can be examined using enzymatic or immunologeic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF‐2) that can be [ 32 P]ADP‐ribosylated by diphtheria toxin and [ 32 P]NAD + . Recognition by the toxin requires the presence in EF‐2 of the unique posttranslationally modifed histidine derivative, dipthamide. The procedure described here reliably discriminates between wild‐type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to faciliating the study of dipthamide biosynthesis in yeast, the more general application of this procedure of this procedure will enable the screening of colonies with assays that require native proteins.