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. 2003 Aug 1;278(31):28470-8.
doi: 10.1074/jbc.M303736200. Epub 2003 May 13.

Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast

Judith W Hyle  1 , Randal J Shaw, Daniel Reines
Affiliations

Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast

Judith W Hyle et al. J Biol Chem. .

Abstract

IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show here that these family members are distinct, despite a high degree of amino acid identity between the proteins they encode. Extrachromosomal copies of IMD1, IMD3, or IMD4 could not rescue the drug-sensitive phenotype of IMD2 deletants. When overexpressed, IMD3 or IMD4 weakly compensated for deletion of IMD2. IMD1 is transcriptionally silent and bears critical amino acid substitutions compared with IMD2 that destroy its function, offering strong evidence that it is a pseudogene. The simultaneous deletion of all four IMD genes was lethal unless growth media were supplemented with guanine. This suggests that there are no other essential functions of the IMPDH homologs aside from IMP dehydrogenase activity. Although neither IMD3 nor IMD4 could confer drug resistance to cells lacking IMD2, either alone was sufficient to confer guanine prototrophy. The special function of IMD2 was provided by its ability to be transcriptionally induced and the probable intrinsic drug resistance of its enzymatic activity.

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Figures

Fig. 1
Fig. 1. Schematic of the IMD gene family of S. cerevisiae
The active site cysteine (*) and specific differences between family members are indicated. Sequence identities were determined by pairwise BLAST analysis (41).
Fig. 2
Fig. 2. Guanine auxotrophy of IMD deletion strains
Cells of each strain (Table I) were grown to saturation in 5 ml of YPD with 0.5 mm guanine and diluted to an initial A600 of 0.01, followed by five consecutive 10-fold dilutions. Five μl of each dilution were spotted onto YPD plates containing 0.5 mm guanine and 10 mm NaOH or 10 mm NaOH (solvent for guanine) alone. Plates were incubated at 30 °C. Yeast strains used were (reading down from the top row) BY4741, DY873, DY874, DY875, DY876, DY877, DY878, DY879, DY880, DY881, DY882, DY883, DY884, DY885, DY886, and DY887.
Fig. 3
Fig. 3. Mycophenolic acid sensitivity of IMD deletants
Yeast strains were grown overnight in YPD with 0.5 mm guanine and diluted to an A600 of 0.01 in SC. Five μl of this suspension and 10-fold serial dilutions thereof were spotted onto SC containing no drug or mycophenolic acid and incubated at 30 °C. Yeast strains used were those indicated in the Fig. 2 legend.
Fig. 4
Fig. 4. Inducibility of each IMD
Yeast strains were grown to an A600 of ≈0.5 in YPD with 0.5 mm guanine. Cells were pelleted, washed with water, and resuspended in SC to an A600 of ≈0.5. Mycophenolic acid (15 μg/ml) was added, and RNA was harvested from aliquots of culture at the indicated times. Northern blots were probed with the indicated IMD genes or SED1 as a control for loading. The strains used were (from left to right) BY4741, DY883, DY884, DY885, DY886, and DY887.
Fig. 5
Fig. 5. Functional complementation of IMD2 deletion by expression of yeast IMD family members
Yeast strains lacking IMD2 and transformed with an empty vector or IMD genes on a single copy (A and B) or 2 μ plasmid (C) were tested for mycophenolic acid resistance. Strains used (Table I) were (reading down from the top row) DY731, DY928, DY942, DY929, DY930, DY931, DY980, DY948, DY981, DY950, DY970, DY731, DY917, DY835, DY918, DY919, DY963, and DY964. Cells in logarithmic growth were diluted to an A600 of 0.025. Ten μl of this and 4-fold serial dilutions thereof were spotted onto SC medium lacking uracil and containing or lacking 7.5 or 15 μg/ml mycophenolic acid and grown at 30 °C. Strains in B contained plasmids in which the respective IMD ORFs were driven by the IMD2 inducible promoter.
Fig. 6
Fig. 6. The guanine response element is required for IMD2 to provide drug resistance
Derivatives of the IMD2 promoter driving the IMD2 ORF (top panels) were tested for their ability to provide drug resistance when transformed into a yeast strain lacking the IMD2 gene. Strains DY964, DY929, DY924, DY926, and DY927 (bottom panels, reading down from the top row) were grown in liquid media with or without mycophenolic acid, diluted, and spotted onto solid media as described in the Fig. 2 legend.
Fig. 7
Fig. 7. Testing of mutant IMD2 derivatives for the ability to provide drug resistance to a strain lacking IMD2
Chimeric and site-directed mutants of IMD2 (A) were transformed into yeast. The resulting strains (Table I) were tested (B) for their ability to grow in the presence and absence of mycophenolic acid as described in the Fig. 2 legend. The strains used were (reading down from the top row) DY970, DY948, DY976, DY977, DY731, DY982, DY983, DY1017, DY1019, and DY985.
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