To discern the specific role of each yeast IMD, it would be ideal to delete the other three copies and study each in isolation. This would provide unequivocal evidence, for example, of whether IMD1 was transcriptionally active in its natural location and whether IMD2 was the only family member that responds to nucleotide pool changes. It was also of interest to generate the quadruple deletant to test for viability and for use as an IMD null that could be reconstituted.
Starting with a diploid yeast strain heterozygous for an IMD3 deletion (Research Genetics), we deleted one allele of each of the remaining three IMD genes by homologous recombination, thereby generating a strain with one intact and one deleted version of each IMD. The resulting diploid was sporulated, and all possible combinations of IMD deletions were recovered (four single deletants, six double deletants, four triple deletants, and one quadruple deletant) based upon their ability to grow on the selective media representing each integrated marker. The genotypes were confirmed by PCR (data not shown). These 15 haploid deletion strains were tested for guanine auxotrophy by assaying for growth on rich medium lacking guanine (). Because the quadruple deletant was unable to grow, we conclude that the family of IMD genes, taken as a group, is essential for yeast viability. In support of the prediction that IMD1 is a pseudogene, the IMD2-IMD3-IMD4 triple deletant also failed to grow, demonstrating that IMD1 is functionally inert in its natural chromosomal context.
Guanine auxotrophy of IMD deletion strains
In organisms with a salvage synthesis pathway, the loss of de novo synthesis of guanine nucleotides can be bypassed by providing guanine in the growth media. The quadruple mutant, as well as the IMD2-IMD3-IMD4 triple deletant, showed wild type growth when the medium was supplemented with guanine (). Neither adenine nor hypoxanthine could rescue growth of the quadruple deletant or the IMD2-IMD3-IMD4 deletant (data not shown). Supplementation with xanthine, the product of IMPDH activity, rescued growth of both strains (data not shown). Hence, loss of the IMD gene family rendered cells auxotrophic for guanine. IMD2, IMD3, or IMD4 rescued growth in the absence of guanine; i.e. each was sufficient to support guanine prototrophy.
We also tested the ability of each deletant to grow in the presence of MPA (). All strains lacking IMD2 were sensitive to MPA. (Because the quadruple deletant and the IMD2-IMD3-IMD4 triple deletant cannot grow without guanine supplementation, and guanine circumvents the drug treatment, these two strains were not informative in this assay.) No combination of other deletions, including the simultaneous deletion of IMD1, IMD3, and IMD4, generated such a phenotype. This formally proved that IMD2 is not functionally equivalent to either IMD1, IMD3, or IMD4 or even to all three of these IMD genes together.
The availability of all four triple deletants provided an opportunity for us to test the transcriptional induction of each IMD in isolation. Cells were grown in liquid media, and RNA was analyzed by Northern blotting at varying times after drug challenge (). Northern blots were probed individually with each of the four IMD genes to ensure that differing hybridization efficiencies did not bias the detection of any family member's mRNA. IMD2 and, to a small extent, IMD4 were induced by mycophenolic acid treatment (, Probe: IMD2, lanes 7–9 and Probe: IMD4, lanes 4–6, respectively). Transcript derived from IMD1 was undetectable (, Probe: IMD1, lanes 10–12). Levels of IMD3 mRNA remained fairly constant throughout the treatment (, Probe: IMD3, lanes 13–15).
Inducibility of each IMD
We used the drug-sensitive phenotype of an IMD2
deletion strain as an assay to test IMD
function. Plasmids bearing IMD1
, or IMD4
and a “reshifted” version of IMD1
, in which a single adenylate residue was deleted to generate a full-length IMD1
reading frame, were introduced into cells. As seen previously (18
), the reintroduction of plasmid-borne IMD2
restored growth on MPA to strains lacking IMD2
(, single copy
). No other IMD
could restore resistance ().
Functional complementation of IMD2 deletion by expression of yeast IMD family members
We next asked whether the unique ability of IMD2 to rescue drug resistance resided in the inducibility of its promoter. We cloned the respective ORFs downstream of the IMD2 promoter and transformed the plasmids into cells lacking IMD2. As expected, episomal IMD2 efficiently restored growth (). Interestingly, IMD3 and IMD4 partially rescued growth when expression of these genes was under control of the IMD2 promoter. Neither IMD1 nor frameshifted IMD1 was effective in restoring growth in equivalent experiments. We confirmed by Northern blot analysis that MPA resulted in transcriptional induction of the each family member under control of the IMD2 promoter (data not shown). This suggested that part, but not all, of the ability of IMD2 to confer MPA resistance was due to transcriptional induction and a concomitant increase in IMPDH enzyme abundance. If so, we reasoned that overexpression of IMD3 or IMD4 by placing these genes with their natural promoters on high copy plasmids might phenocopy the observations seen in . When present on high copy plasmids, IMD3 and IMD4 again provided partial relief from drug inhibition (). We conclude that during drug challenge, the IMD2 gene product is both quantitatively and qualitatively unique among the family of IMPDHs in yeast because part of the cellular response involves making more IMPDH enzyme, and even when overexpressed, IMD3 and IMD4 were only partially active in providing function.
Prior studies have identified regions of the IMD2
promoter that govern the IMD2
transcriptional response in reporter as-says (16
). These include a GRE ≈300 bp upstream of the transcription start site and a RE surrounding the transcription start site (). To test whether these elements were important for cell growth and drug resistance, we introduced into cells plasmids with derivatives of the IMD2
promoter driving transcription of the IMD2
ORF (). A copy of IMD2
lacking all promoter sequences upstream of the transcription start site did not confer MPA resistance (, pIMD2-GRE,RE
). A construct lacking the GRE but containing the RE was virtually inactive (, pIMD2-GRE
). A construct deleted for the RE, whose absence de-represses basal levels of IMD2
transcription but does not affect induction, was fully competent to rescue growth (, pIMD2-RE
). Hence, the transcriptional control sequences identified in reporter assays demonstrated the expected behavior in the MPA bioassay, confirming the importance of transcriptional induction in the acquisition of drug resistance.
The guanine response element is required for IMD2 to provide drug resistance
IMD2 and IMD1 differ by only 20 amino acids. Yet “re-shifted” IMD1 was completely inactive in these assays, even when it was overexpressed. This suggested that the putative transcriptional silencing of IMD1 due to its telomere-proximal location is insufficient to explain its inactivity in vivo. Presumably, 1 or more of the 20 amino acid substitutions inactivated the IMD1 protein. We tested this idea using chimeric constructs made by exchanging restriction fragments between the IMD1 and IMD2 ORFs (). These chimeric reading frames were placed downstream of the IMD2 promoter, introduced into an IMD2 deletant, and tested for their ability to confer MPA sensitivity to transformants. Substituting the first 184 amino acids of IMD1 for those of IMD2 inactivated the ability of IMD2 to provide drug resistance (, construct 3; ). Substituting the carboxyl-terminal 339 amino acids of IMD2 with those from IMD1 also inactivated IMD2 (, construct 4). Hence, there are substitutions in both the amino-terminal third and carboxyl terminal two-thirds of IMD1 that contribute to its inactivity. The 184 amino-terminal residues of IMD1 were independently introduced into IMD2 in two segments, as residues 1–106 or residues 107–184 (, constructs 5 and 6, respectively). Substitution of the amino-terminal 106 amino acids of IMD1 completely inactivated IMD2 (, construct 5). Exchanging the segment from 107–184 severely compromised its function (, construct 6). The latter finding indicates there is yet a third change in the IMD1 sequence that negatively impacts its function in this assay. The region from 1–106 contains three amino acid differences between IMD1 and IMD2. At two of these positions, IMD1 varies from the three other IMD genes that have the same amino acid. We independently engineered both substitutions into the reading frame of IMD2 by site-directed mutagenesis to test whether either was sufficient to inactivate IMD2 (). The G93D substitution (
construct 8) completely inactivated IMD2, whereas the T38A change (construct 7) had little effect. Thus, we have identified one of the amino acid changes that accounts for the biological inactivity of IMD1. The reciprocal change (D93G) introduced into IMD1 did not reactivate it (data not shown). Taken together, these data indicate that IMD1 possesses numerous amino acid substitutions that inactivate its ORF.
Testing of mutant IMD2 derivatives for the ability to provide drug resistance to a strain lacking IMD2
Recent reports have described point mutations in a human IMPDH
that may be causally involved in the inheritance of retinitis pigmentosa (5
). One such mutation is a substitution of asparagine for an aspartate that is conserved between human IMPDH1
, and all four yeast IMD
genes. This change was predicted to be highly deleterious to IMPDH function (5
). The mycophenolic acid sensitivity assay allowed us to test whether the comparable D → N change in yeast IMD2
compromised its function. The D229N change did not reduce the ability of IMD2
to provide guanine auxotrophy (data not shown) or confer MPA resistance to yeast ().