Yeast Yeast 2003; 20: 39–51. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.933 Research Article Analysis of the hypoxia-induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast Volkmar Passoth 1,2 *, Marita Cohn 3 , Bernd Sch¨ afer 4 , B¨ arbel Hahn-H¨ agerdal 1 and Ulrich Klinner 4 1 Applied Microbiology, Lund University, PO Box 124, SE-221 00 Lund, Sweden 2 Klinik f ¨ ur innere Medizin B, Ernst-Moritz-Arndt-Universit¨ at Greifswald, F.-Loeffler-Strasse 23a, D-17487 Greifswald, Germany 3 Department of Cell and Organism Biology (Molecular Genetics), Lund University, S¨ olvegatan 35, S-223 62 Lund, Sweden 4 Institut f ¨ ur Biologie IV (Mikrobiologie), RWTH Aachen, Worringer Weg, D-56056 Aachen, Germany *Correspondence to: Volkmar Passoth, Swedish University of Agricultural Sciences (SLU), Department of Microbiology, Uppsala, Genetic Center, Box 7025, SE-750 07 Uppsala, Sweden. E-mail: Volkmar.Passoth@mikrob.slu.se Received: 12 July 2002 Accepted: 16 September 2002 Abstract We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co 2+ . Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co 2+ but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast. Copyright  2002 John Wiley & Sons, Ltd. Keywords: oxygen limitation; hypoxia; alcohol dehydrogenase; green fluorescent protein; promoter analysis Introduction Pichia stipitis is a respiratory yeast and the regu- lation of its aerobic and fermentative metabolism is different from that of the model organism of yeast physiology, Saccharomyces cerevisiae. The enzymes of the fermentative pathway, pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are regulated independently of the gly- colytic flux (Passoth et al., 1996). There are two ADH-genes cloned from P. stipitis. One of them, PsADH2, has been shown to be induced under oxy- gen limitation on the transcriptional level (Passoth et al., 1998; Cho and Jeffries, 1999). The nature of the signal that leads to hypoxic ADH induction is not yet known. Lack of oxygen leads to significant transcrip- tional up- or downregulation of about 360 genes of baker’s yeast, S. cerevisiae (Ter Linde et al., 1999). Several systems of oxygen sensing are known in S. cerevisiae (Zitomer et al., 1997; Kwast et al., 1998). The one investigated best is the HAP /ROX system. In this regulatory system a variety of aero- bic genes are regulated by the transcriptional factor HAP1p. Heme is an essential co-factor for the func- tion of HAP1p and molecular oxygen is essential for the synthesis of heme (Labbe-Bois and Labbe, 1990). If there is a shortage of oxygen, the intracel- lular level of heme decreases and the activation of HAP1p-regulated genes fails. Among the HAP1p- regulated aerobic genes there is also ROX1, which encodes a transcriptional repressor. This repressor Copyright  2002 John Wiley & Sons, Ltd.