Basic helix-loop-helix transcription factor heterocomplex of Yas1p and Yas2p regulates cytochrome P450 expression in response to alkanes in the yeast Yarrowia lipolytica

Abstract

The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica, is highly regulated and can be induced by alkanes. Previously, we identified a cis-acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.

FIG. 1.

Relationship of YALI0E32417g/Yas2p with other bHLH proteins. (A) Alignment of the bHLH motifs. Sequences were aligned by the CLUSTALW program (45) on the website of the Kyoto University Bioinformatics Center by using the slow/accurate mode and default parameters (http://clustalw.genome.jp/). Amino acids identical among the six proteins are shown by asterisks, and conserved amino acids are indicated by dots. (B) Phylogenetic relatedness of the YALI0E32417g/Yas2p bHLH motif to other bHLH proteins. Shown is a dendrogram reconstructed using CLUSTALW methods (http://clustalw.genome.jp/). (C) Schematic diagrams of bHLH proteins. The bHLH motifs are indicated by gray boxes. The glutamine-rich (Q rich) region and the potential nuclear localization signal (NLS) are shown as striped and filled boxes, respectively. The first two letters in the protein labels indicate the species of origin: Yl, Y. lipolytica; Sc, S. cerevisiae; Dh, D. hansenii; Hs, Homo sapiens. Swiss-Prot accession numbers are as follows: S. cerevisiae Ino4p, P13902; S. cerevisiae Ino2p, P26798; S. cerevisiae Cbf1p, P17106; Homo sapiens SREBP1, P36956; and Homo sapiens AhR, P35869. For Y. lipolytica and D. hansenii ORF products, ordered locus names as designated in the Génolevures project are indicated. See the text for discussion about DEHA0B04741g and DEHA0D19877g.

FIG. 2.

Requirement of the YAS2 gene for alkane-dependent transcription induction and growth on alkanes. (A) ALK1 gene expression was not induced by n-decane and n-hexadecane in the YAS2 deletion mutant. Cells were grown in glycerol-containing medium, transferred to minimal media containing glycerol, glucose, n-decane, n-hexadecane, or oleic acid, and cultured for 1 h. One microgram of total RNA was loaded. WT, wild type; EtBr, ethidium bromide. (B) The defect in alkane-responsive transcription induction in the YAS2 deletion mutant was abolished by the introduction of the YAS2-carrying plasmid pSYAS2. pSUT5 is an empty vector. Culture and RNA loading conditions are described in the legend to panel A. (C) The YAS2 deletion mutant carrying vector pSUT5 did not grow on n-decane and n-hexadecane, but pSYAS2 restored growth. (D) The YAS2 deletion mutant did not grow in liquid medium with n-decane, but it did grow with oleic acid. Two percent glucose or 1% oleic acid was added to minimal medium. Two percent n-decane was added initially, followed by supplementation with 1% n-decane every 24 h. OD₆₆₀, optical density at 660 nm.

FIG. 3.

Inositol prototrophy of yas1Δ and yas2Δ strains. The growth of yas1Δ and yas2Δ mutants in the presence (inositol +) or absence (inositol −) of inositol was compared to that of the wild-type CXAU/A1 strain. S. cerevisiae wild-type (Sc WT) and inositol auxotroph mutant (Sc ino-) strains were used as controls.

FIG. 4.

Transcriptional activation by Yas2p. (A) Schematic diagrams of Gal4BD fusion proteins. The pBD-GAL4 Cam phagemid vector (pBD) producing Gal4BD, pBD-YAS1 producing the Gal4BD-Yas1 fusion protein, or pBD-YAS2 producing the Gal4BD-Yas2 fusion protein was introduced into S. cerevisiae YRG-2. (B) Yas2p has a transactivation function. Transcriptional activation of the reporter genes UAS_GAL1-TATA_GAL1-HIS3 and UAS_GAL4-TATA_CYC1-lacZ was detected by using a plate containing 10 mM 3-AT (−His + 3-AT) and by using a β-galactosidase assay (+His), respectively. The results shown are the averages ± the standard errors of the means of results from six independent experiments.

FIG. 5.

Interaction between Yas1p and Yas2p. (A) Positive interaction between Yas1p and Yas2p in a yeast two-hybrid assay. Bait plasmids pBD-GAL4 Cam (pBD) and pBD-YAS1 are described in the legend to Fig. 4. Prey plasmids pAD-GAL4-2.1 phagemid vector (pAD), producing Gal4AD, and pAD-YAS2, producing the Gal4AD-Yas2 fusion protein, were introduced into S. cerevisiae YRG-2. Specific interaction was detected by assessing the activation of the reporter genes, UAS_GAL1-TATA_GAL1-HIS3 and UAS_GAL4-TATA_CYC1-lacZ, by using a 3-AT-containing plate (−His + 3-AT) and β-galactosidase activity (+His), respectively. The results shown are the averages ± the standard errors of the means of results from six independent experiments. (B) Specific interaction of recombinant Yas1p and Yas2p in vitro. Bacterially expressed GST or GST fusion proteins were immobilized on glutathione-Sepharose beads and then incubated with extracts containing His-tagged Yas1p or Yas2p. After washing of the beads, bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting (IB). Immunoblotting with anti-His tag antibody detected the presence of His₆-Yas1p (left upper panel) or His₆-Yas2p (right upper panel). Immunoblotting with anti-GST antibody demonstrated the binding of GST, GST-Yas1p, and GST-Yas2p to the beads (lower panels).

FIG. 6.

Formation of Yas1p-Yas2p complex on ARE1. (A) The nucleotide sequence of a probe used for the electrophoretic mobility shift assay. ARE1 (CTTGTGNXCATGTG) is underlined. (B) Binding of the Yas1p-Yas2p heterocomplex to ARE1. Bacterial cell extracts containing GST, GST-Yas1p, or His₆-Yas2p were incubated with ³²P-labeled probe. The DNA-protein complex was resolved on a 5% polyacrylamide gel. +, present; −, absent.

FIG. 7.

Model for the alkane-dependent transcription induction by the Yas1p-Yas2p heterocomplex. The potential bHLH heterodimer complex of Yas1p and Yas2p binds to ARE1 (CTTGTGN_XCATGTG) in the ALK1 promoter, and it induces the transcription of ALK1 in the presence of alkanes. The Yas1p-Yas2p complex also binds the promoters of other genes involved in the alkane utilization, including the acetoacetyl-CoA thiolase gene and the YAS1 gene itself. The positive autoregulatory feedback loop of Yas1p makes it possible to induce massive changes in gene expression in response to alkanes and to adapt quickly to exposure to alkanes.