Abstract
Environmental pH exerts broad control over growth and differentiation, but the molecular responses to external pH changes are poorly understood. Here we have used open reading frame macroarray hybridization to identify alkaline response genes in Saccharomyces cerevisiae. Northern or lacZ fusion assays confirmed the alkaline induction of two ion pump genes (ENA1 and VMA4), several ion limitation genes (CTR3, FRE1, PHO11/12, and PHO84), a siderophore-iron transporter gene (ARN4/ENB1), two transcription factor genes (NRG2 and TIS11), and two predicted membrane protein genes (YAR068W/YHR214W and YOL154W). Unlike ENA1 and SHC1, these new alkaline response genes are not induced by high salinity. The known pH-responsive genes in other fungi depend on the conserved PacC/Rim101p transcription factor, but induction of several of these new genes relied upon Rim101p-independent pH signaling mechanisms. Rim101p-dependent genes were also dependent on Rim13p, a protease required for Rim101p processing. The Rim101p-dependent gene VMA4 is required for growth in alkaline conditions, illustrating how Rim101p may control adaptation. Because Rim101p activates ion pump genes, we tested the role of RIM101 in ion homeostasis and found that RIM101 promotes resistance to elevated cation concentrations. Thus, gene expression surveys can reveal new functions for characterized transcription factors in addition to uncovering physiological responses to environmental conditions.
Highlights
Extracellular pH is a key environmental signal that influences growth, physiology, and differentiation [1,2,3,4,5,6,7,8,9]
These genes included ENA1, which is induced at pH 8 [14, 18], but not SHC1 or SCY1, which are induced at higher pH [19]
Fungal Rim101p/PacC homologs are known regulators of nonessential pH response genes; we extend this understanding with the identification of a Rim101pdependent gene that is essential for alkaline adaptation, with identification of Rim101p-independent alkaline response genes, and with the finding that Rim101p is required for general ion homeostasis
Summary
Extracellular pH is a key environmental signal that influences growth, physiology, and differentiation [1,2,3,4,5,6,7,8,9]. The vacuolar ATPase is required for vacuolar acidification, which cannot occur through endocytosis in alkaline media [16, 17] Null mutants lacking these pumps are hypersensitive to alkaline growth conditions and elevated external cation concentrations but are capable of growth in acidic media. Alkaline pH induces transcription of IPNA and other penicillin biosynthetic genes in Aspergillus nidulans and Penicillium chrysogenum [20, 21], the protease gene XPR2 in Yarrowia lipolytica [22], and cell wall protein genes in Candida albicans [23,24,25] These responses depend upon a conserved signal transduction pathway (20, 22, 26 –29) that is best understood from work on the A. nidulans transcription factor PacC. Their sole known function is to promote PacC cleavage, because loss-of-function pal mutations cause similar phenotypes to loss-of-function pacC mutations and because expression of C-terminally truncated PacC derivatives suppresses pal mutant defects [30, 31]
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