Genetics. Published Articles Ahead of Print: October 20, 2008, Copyright © 2008
doi:10.1534/genetics.108.094458


A more recent version of this article appeared on December 1, 2008.

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

Andrea Spitzner 1, Angelika F. Perzlmaier 2, Kerstin E. Geillinger 3, Petra Reihl 4 and Juergen Stolz 3*

1 Universität Stuttgart
2 Universität Regensburg
3 Technische Universität München
4 Landesbetrieb Hessisches Landeslabor, Kassel

* To whom correspondence should be addressed. E-mail: stolz{at}wzw.tum.de.

Submitted on July 28, 2008
Revised on August 12, 2008
Accepted on 20 October 2008


Abstract

Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B2). 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 over-express the plasma-membrane riboflavin transporter MCH5. Frequently, this over-expression is mediated by the transcription factor Put3 that 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.

Key Words: MCH5, PUT3, RIB5, proline utilization, riboflavin biosynthesis and transport