FKBP12 Controls Aspartate Pathway Flux in Saccharomyces cerevisiae To Prevent Toxic Intermediate Accumulation

Abstract

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate ␤-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate ␤-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.Prolyl isomerases are widely conserved, ubiquitous enzymes that catalyze cis-trans isomerization of peptidyl-prolyl bonds, a reaction that can be rate limiting for protein folding. Founding members of this group of enzymes are cyclophilin A, previously identified as the cyclosporine A receptor (25,31,75), and the structurally unrelated enzyme FK506 binding protein FKBP12 (35). A third family of prolyl isomerases, known as the parvulins, was discovered for bacteria (60, 61) and later was found to be conserved in many other organisms.Cyclophilin A and FKBP12 mediate the immunosuppressive effects of cyclosporine A and FK506 in mammals by forming complexes with these drugs that bind to and inhibit the functions of calcineurin in T-cell activation (for a review, see reference 68). FKBP12 is also the receptor for the drug rapamycin, and the FKBP12-rapamycin complex inhibits the functions of the Tor proteins (36, 44). Both cyclophilin A and FKBP12 are conserved in budding yeast (where they are encoded by the CPR1 and FPR1 genes, respectively) and mediate calcineurin inhibition by cyclosporine A and FK506 (11,26,30,37,38,52,55,78,83) and Tor inhibition by rapamycin (36, 44). Saccharomyces cerevisiae expresses seven other cyclophilins (Cpr2 to Cpr8), three other FKBPs (Fpr2 to Fpr4), and a single parvulin (Ess1) (for a recent review, see reference 6).With the exception of Ess1, all yeast prolyl isomerases are dispensable for growth (18,32,34). However, cyclophilin A does become essential in cells compromised for Ess1 function, suggesting a functional overlap between these two structurally unrelated prolyl isomerases (4). Thus far, the endogenous functions that have been defined for these prot...

Identification of Genes Required for Protection from Doxorubicin by a Genome-Wide Screen in Saccharomyces cerevisiae

“…Inactivation of the HOM6 gene, which encodes the homoserine dehydrogenase, results in accumulation of L-aspartate-semialdehyde, a precursor of homoserine. L-Aspartate-semialdehyde has been shown to be toxic in certain genetic backgrounds (45). Interestingly, inhibition of the subsequent steps of the reaction also results in sensitivity to doxorubicin.…”

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

“…An aspect of complexity is that aspartate semialdehyde is an unstable compound that polymerizes in solution (79). It has also been reported to be quite toxic (3). Thus, on one hand, the multiple enzymes competing for aspartate semialdehyde must access a substrate available at low concentrations and vulnerable to short half-life conditions, but on the other hand, regulatory restraints on the enzymes using the semialdehyde must not result in undue accumulation.…”

The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit

“…As such, isomerases participate in the coordination of the cell cycle (Lu et al 1996;Yeh and Means 2007) and transcription (Nelson et al 2006;Wang et al 2010;Dilworth et al 2012), which is presumably accomplished through the recruitment to, and isomerization of, substrate proteins. However, there are examples in which this is not the case, and prolyl isomerases function independent of catalytic activity (Arévalo-Rodríguez et al 2004;Riggs et al 2007). Of the 18 annotated FKBP prolyl isomerases in humans, few have been extensively characterized, limiting the resolution of FKBP interactions, substrates, and biological functions.…”