Copyright Ó 2008 by the Genetics Society of America
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 Reihl4 and Jurgen Stolz5
Lehrstuhl fur Zellbiologie und Pﬂanzenphysiologie, Universitat Regensburg, Universitatsstraße 31, 93040 Regensburg, Germany
¨ ¨ ¨
Manuscript received July 28, 2008
Accepted for publication October 20, 2008
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
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
M AMMALS depend on a dietary supply of riboﬂavin
(vitamin B2), which mostly derives from the
ﬂavoprotein cofactors FMN and FAD. These are de-
riboﬂavin and are used in industrial processes for ribo-
ﬂavin synthesis (Stahmann et al. 2000).
In addition to being able to synthesize riboﬂavin, single-
adenylated or dephosphorylated in the gut followed by celled organisms are also capable of taking up riboﬂavin
the transport of free riboﬂavin across the mucosal mem- from the culture medium. Because the riboﬂavin trans-
brane (Foraker et al. 2003). In contrast, although many port activities of most wild-type (wt) strains are low, most
microorganisms are dependent on various water-soluble investigations were performed with riboﬂavin auxotro-
vitamins, only few show a riboﬂavin auxotrophy (Koser phic mutants. At least three different classes of riboﬂavin
1968). This indicates that most microorganisms are transporters exist in bacteria. These have been pre-
capable of synthesizing riboﬂavin, a pathway, which starts dicted by phylogenetic footprinting (Vitreschak et al.
with GTP and two molecules of ribulose-5-phosphate and 2002) and functional data are now available for two
is similar but not perfectly conserved in various species proteins. RibU from Lactococcus lactis and Bacillus subtilis
(Bacher et al. 2000). In the yeast Saccharomyces cerevisiae, appear to work as very high-afﬁnity transporters with ﬁve
which is an excellent dietary source of riboﬂavin transmembrane domains (Cecchini et al. 1979; Burgess
(Bassler et al. 2002), the enzymes required for riboﬂavin
¨ et al. 2006; Vogl et al. 2007). According to our analyses,
synthesis are encoded by the genes RIB1, RIB2, RIB3, RibU acts as an active riboﬂavin transporter in B. subtilis.
RIB4, RIB5, and RIB7. Both prokaryotic and eukaryotic Proteins of the RibM type are present in Corynebacterium
microorganisms have been engineered to overproduce glutamicum and Streptomyces davawensis (Grill et al. 2007;
Vogl et al. 2007) and RibM from C. glutamicum acts as a
facilitator when expressed in Escherichia coli. The third
Present address: Institut fu Biochemie, Universitat Stuttgart, Pfaffen-
¨r ¨ prototype bacterial riboﬂavin transporter, ImpX, has
waldring 55, 70569 Stuttgart, Germany. not been experimentally studied (Vitreschak et al.
Present address: Lehrstuhl fu Genetik, Universitat Regensburg, Uni-
¨r ¨ 2002). Yet another type of plasma-membrane riboﬂavin
versitatsstraße 31, 93040 Regensburg, Germany.
transporter is present in fungi. We used a multicopy
Present address: Lehrstuhl fu Ernahrungsphysiologie, Technische
¨ ¨nchen, Wissenschaftszentrum Weihenstephan, Am Forum suppressor screen of S. cerevisiae riboﬂavin auxotrophic
5, 85350 Freising-Weihenstephan, Germany. strains to identify MCH5, the ﬁrst known eukaryotic
Present address: Landesbetrieb Hessisches Landeslabor, Standort riboﬂavin transporter gene (Reihl and Stolz 2005).
Kassel, Druseltalstraße 67, 34131 Kassel, Germany.
Riboﬂavin transport in yeast is not signiﬁcantly stimu-
Corresponding author: Lehrstuhl fu Ernahrungsphysiologie, Technische