Screening the Yeast “Disruptome” for Mutants Affecting Resistance to the Immunosuppressive Drug, Mycophenolic Acid

Christine Desmoucelles,Benoı̂T Pinson,Christelle Saint-Marc,Bertrand Daignan-Fornier

doi:10.1074/jbc.m111433200

Abstract

The immunosuppressive drug mycophenolic acid (MPA) is a potent and specific inhibitor of IMP dehydrogenase, the first committed step of GMP synthesis. A screen for yeast genes affecting MPA sensitivity, when overexpressed, allowed us to identify two genes, IMD2 and TPO1, encoding a homologue of IMP dehydrogenase and a vacuolar pump, respectively. In parallel, 4787 yeast strains, each carrying an identified knock-out mutation, were tested for growth in the presence of MPA, allowing identification of 100 new genes affecting MPA resistance when disrupted. Disturbance of several cellular processes, such as ergosterol biosynthesis, vacuole biogenesis, or glycosylation impaired the natural capacity of yeast to resist MPA, although most of the highly sensitive mutants affected the transcription machinery (19 mutants). Expression of TPO1 and/or IMD2 was strongly affected in 16 such transcription mutants suggesting that low expression of these genes could contribute to MPA sensitivity. Interestingly, the spt3, spt8, and spt20 mutants behaved differently than other Spt-Ada-Gcn5-acetyltransferase (SAGA) mutants. Indeed, in these three mutants, as in previously characterized transcription elongation mutants, IMD2 expression was only affected in the presence of MPA, thus suggesting a possible role for some SAGA subunits in transcription elongation.

Highlights

Increase yeast sensitivity to mycophenolic acid (MPA) had been identified previously; these affect proteins such as TFIIS, Rpb2p, Rtf1p, Rpb9p, or Paf1p that are required for optimal transcription elongation [3,4,5,6,7]
Plasmids from three yeast MPA resistant transformants were purified in Escherichia coli and shown to confer MPA resistance when retransformed in yeast
Sequence of the plasmid inserts revealed that they all contained a fragment of chromosome VIII carrying the IMD2 gene encoding an isoform of IMP dehydrogenase, the enzyme inhibited by MPA

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains—The following yeast strains were used: Y350 (MAT␣; ura; leu112; lys2⌬201), BY4742 (MAT␣; his3⌬1; leu2⌬0; lys2⌬0; ura3⌬0). An ApaI 5.06-kpb fragment containing the IMD2 gene was deleted from the original P1736 plasmid. To construct P1829, a 7-kpb BbuI restriction fragment was deleted from plasmid P1736. P1937 was constructed by introducing a kanamycin-resistance cassette in the IMD2 gene at the unique BamH1 restriction site. Subcloning of the plasmid carrying the TPO1 region was done as follows. The TPO1 gene was amplified by PCR from yeast genomic DNA and cloned in YEpLac195 (URA3, 2 ␮m) [10] using the BamH1 and HindIII restriction sites in the primer sequences. Blots were prepared and probed [11] with labeled PCR fragments, amplified from the S288C genomic DNA template with the following pairs of oligonucleotides: IMD2, 155, 5Ј-TTTTTGCATGCCGCCATTAGAGACTAC-3Ј, and 19, 5Ј-TCCCCCGGGCCCAAATACCGTA-3Ј; TPO1, 453, 5Ј-CGCGGATCCTACTCTTGTGCTTGTGCATAG-3Ј, and 454, 5Ј-TAACCAGGCACCAGTAGTACT-3Ј; PDA1, 575, 5Ј-TGTCGCACCTGTATCTTCAC-3Ј, and 576, 5Ј-AGCGTAAAGGTGCATGGAAC-3Ј. Quantification of radioactive spots was performed using a PhosphorImager and the ImageQuant analysis software (Amersham Biosciences)

RESULTS AND DISCUSSION

Cell cycle and cytoskeleton

To MPA

Regulator of meiosis and spore formation