The Proline-Dependent Transcription Factor Put3 Regulates the Expression of the Riboflavin Transporter MCH5 in Saccharomyces cerevisiae

Abstract

Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B 2 ). Riboflavin auxotrophic mutants with deletions in any of the RIB genes frequently segregate colonies with improved growth. We demonstrate by reporter assays and Western blots that these suppressor mutants overexpress the plasma-membrane riboflavin transporter MCH5. Frequently, this overexpression is mediated by the transcription factor Put3, which also regulates the proline catabolic genes PUT1 and PUT2. The increased expression of MCH5 may increase the concentrations of FAD, which is the coenzyme required for the activity of proline oxidase, encoded by PUT1. Thus, Put3 regulates proline oxidase activity by synchronizing the biosynthesis of the apoenzyme and the coenzyme FAD. Put3 is known to bind to the promoters of PUT1 and PUT2 constitutively, and we demonstrate by gel-shift assays that it also binds to the promoter of MCH5. Put3-mediated transcriptional activation requires proline as an inducer. We find that the increased activity of Put3 in one of the suppressor mutants is caused by increased intracellular levels of proline. Alternative PUT3-dependent and -independent mechanisms might operate in other suppressed strains.

Put3 Positively Regulates Proline Utilization in Candida albicans

“…The key proline catabolism enzyme-encoding genes PUT1 and PUT2 ( Table 2 ) were among the genes upregulated in strains expressing the hyperactive Put3. As well, the C. albicans gene orf19.1584, which is similar to the gene MCH5 encoding a riboflavin transporter required for Put1 function in proline degradation in S. cerevisiae ( 12 ), was also upregulated. This suggests that Put3 has preserved its proline catabolism regulation role in C. albicans and S. cerevisiae , and it regulates this pathway by transcriptionally activating PUT1 and PUT2 , as well as the Put1 cofactor precursor importer gene, orf19.1584.…”

“…Consistent with our observations from phenotypic studies and RNA-seq data, both PUT1 and PUT2 in C. albicans are under the regulation of Put3, as shown by our transcriptional profiling data, and our ChIP-chip data suggest that Put3 binds the PUT1 promoter ( Table 2 ), while PUT2 does not appear to be bound by Put3 in C. albicans . MCH5 is also a direct target of Put3 in S. cerevisiae and encodes a riboflavin transporter ( 12 ). Riboflavin is required for the generation of flavin adenine dinucleotide (FAD), the catalytic cofactor required for Put1 activity ( 12 ).…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…Nevertheless, other work has discussed the role that the RF transporter Mcf5p plays in facilitating RF flux into and out of the cell (364). Activation of the transcription factor encoded by PUT3 is involved in adapting S. cerevisiae RF auxotrophs to low concentrations of exogenous RF (460).…”

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis

“…Mining of protein-protein interaction and genome databases uncovered strong evidence that such a complex exists in diverse organisms (Supplementary Figure S5A). Thus, in microbes, protein-protein interactions have been detected biochemically [28–32] or genetically [33] between each of the six riboflavin biosynthesis enzymes and at least one of the others. Second, sequenced genomes encode pairwise fusions between almost all of the riboflavin enzymes and triple fusions among several of them [34], which proves their potential to physically associate.…”