Roles of ATR1 paralogs YMR279c and YOR378w in boron stress tolerance

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Abstract

Boron is a necessary nutrient for plants and animals, however excess of it causes toxicity. Previously, Atr1 and Arabidopsis Bor1 homolog were identified as the boron efflux pump in yeast, which lower the cytosolic boron concentration and help cells to survive in the presence of toxic amount of boron. In this study, we analyzed ATR1 paralogs, YMR279c and YOR378w, to understand whether they participate in boron stress tolerance in yeast. Even though these genes share homology with ATR1, neither their deletion rendered cells boron sensitive nor their expression was significantly upregulated by boron treatment. However, expression of YMR279, but not YOR378w, from the constitutive GAPDH promoter on a high copy plasmid provided remarkable boron resistance by decreasing intracellular boron levels. Thus our results suggest the presence of a third boron exporter, YMR279c, which functions similar to ATR1 and provides boron resistance in yeast.

Highlights

ATR1 paralog YMR279c plays role in boron detoxification. ► YMR279c overexpression lowers cytoplasmic boron levels. ► ATR1 paralog YOR378w has no roles in boron stress response.

Introduction

Boron is an essential micronutrient for various species [1], [2], [3], [4]. The essentiality of boron is known almost for 90 years for plants [5]. In plants, boron act as a cross-linker of rhamnogalacturonan-II molecule [6] within the cell wall and maintains stability of the plant cell structure, and its deficiency has been linked to growth problems in Arabidopsis thaliana [4].

A. thaliana Bor1 is the first identified boron transporter, functions as an efflux-pump and essential when limited amount of boron is available for growth [7]. Yeast Bor1 has been identified as a boron exporter having role in boron tolerance [8]. In a recent study, Atr1 was suggested as the main boron exporter in yeast [9]. Atr1 deficient cells were boron sensitive and accumulated higher level of intracellular boron, whereas ATR1 overexpressing cells were resistant to toxic amount of boron application and contained less boron. ATR1 transcription was induced several fold by boron treatment [9].

Yeast ATR1 belongs to the DHA2 family of drug: H+ antiporters with 10 members in the budding yeast, and this transporter family is conserved among yeast species such as Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya gossypii [10]. Seven of DHA2 family members including YMR279C and YOR378W have not yet been characterized [11]. Since YMR279C and YOR378W are the two closest paralogs of ATR1, we speculated that they may play a role in boron stress response. To test this idea, both genes were cloned and overexpressed in yeast. Expression of YMR279C, but not YOR378W, provided boron resistance by decreasing intracellular boron level.

Section snippets

Yeast growth and media

WT strain BY4741 (MATa his3 leu2 met15 ura3) and its isogenic atr1Δ, ymr279cΔ, and yor378wΔ deletion mutants were obtained from the yeast deletion library (Invitrogen). YPD (2% glucose, 2% peptone, 1% yeast extract, and 2% agar for solid media) and YNB media (0,67% yeast nitrogen base without amino acids, 2% glucose, 2% agar for solid media) were used for cell growth. For spotting assays, overnight cultures were diluted to an optical density of 0.2, 0.02, 0002, and 0.0002 at 600 nm by serial

Boron effects on cell growth

Cells lacking YMR279c and YOR378w genes were previously tested for boron sensitivity and they were found to be not boron sensitive [9]. Based on this observation, both YMR279c and YOR378w genes have not been considered for further investigation for their roles in boron tolerance. However, when their primary structures are considered, Ymr279c and Yor378w show 65% and 40% homology to Atr1, respectively. The N-terminal region is the most heterogenous part in all three proteins (Fig. S1). In spite

Discussion

In yeast, mechanisms of boron resistance are centered around exportation of boron out of cells. Two membrane proteins, Atr1 and Bor1, were previously characterized as membrane boron efflux transporters [9], [18]. Here we showed that Ymr279c, one of the two paralogs of Atr1, provides boron resistance by decreasing intracellular boron concentrations similar to Atr1. However, there are differences between Atr1 and Ymr279c roles in boron tolerance. YMR279c is slightly upregulated by boron at the

Acknowledgments

We thank to IYTE-Biotechnology center and IYTE-Biological mass spectrometry facilities for some instrumental help. This work was supported by TUBITAK 108T181 and TUBA-GEBIP Grants to A.K.

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