Mutants of the eukaryote Saccharomyces cerevisioe, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL) promoter element in a plasmid.Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GALI promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or a-melanocytestimulating hormone along with the lipid-associating domains of fragment B and the leader peptide ofthe Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.The lethal action of diphtheria toxin on susceptible cells is mediated by the inhibition of protein synthesis through the ADP-ribosylation of elongation factor 2 (EF-2) (1). A single molecule of the toxin in the cytoplasm is sufficient to kill mammalian cells (2). This toxin-catalyzed activity is specific for EF-2 and occurs at a unique posttranslational histidine derivative, diphthamide (3, 4), found in a conserved amino acid sequence (5) in the EF-2 of all eukaryotes and archaeobacteria examined thus far (6). Rare eukaryotic mutant cell lines defective in this posttranslational modification possess EF-2 that cannot serve as a substrate for diphtheria toxin, and they are thus resistant to its cytotoxic effects (7). Because of the profound cytotoxicity, well-characterized mode of action, and defined mechanism of cellular transport of diphtheria toxin, its fragments have been employed in the construction of hybrid toxins. These conjugate toxins have been assembled by the chemical modification and disulfide linkage of the toxin catalytic fragment with cell-surfacedirected agents such as monoclonal antibodies and peptide hormones. Although target-specific toxicity has been demonstrated (8-12), the stoichiometry and sites of linkage of such conjugates are heterogeneous, creating complications for their application and confounding the interpretation of experimental results.To obviate these problems, fusion genes ofdiphtheria toxin and a-melanocyte-stimulating hormone (a-MSH) or diphtheria toxin and interleukin 2 (IL-2) have recently been constructed (13)(14)(15) The eukaryote Saccharomyces cerevisiae has proven to be of particular utility for the cloning and expression of heterologous proteins because it is readily amenable to biochemical manipulation and genetic engineering to avoid proteolysis and to enhance yield. We recently reported a selection procedure for mutants of S. cerevisiae defective in the synthesis of diphthamide and conseq...

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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.

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