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HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH

Vanda Carmelo, Isabel Sá-Correia
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb10312.x 85-88 First published online: 1 April 1997


During exponential growth of Saccharomyces cerevisiae at the inhibitory pH 2.5, the transcription of the major small-heat-shock-protein-encoding gene HSP26 was strongly induced while at the optimal pH 5.0, the mRNA levels from the HSP26 gene were undetectable. When yeast cells entered the stationary phase of growth at pH 5.0, transcription was dramatically enhanced and the level of the HSP26 transcripts reached similar values in stationary cells grown at optimal or inhibitory low pH.

  • Saccharomyces cerevisiae
  • HSP26 gene transcription
  • Acid stress

1 Introduction

In response to adverse environmental conditions, namely heat shock, all cells co-ordinately induce the synthesis of a set of proteins, the heat shock proteins (Hsps). Among them, Hsp26 is a member of the low molecular mass hsp family in yeast [15]. This protein is not present in unstressed vegetative cells but is strongly induced by heat shock, stationary phase arrest or nitrogen starvation [1, 2, 5] and salt shock [6]. The cellular role of Hsp26 remains to be elucidated, however, the gene is not essential for the acquisition of thermotolerance in exponential or in stationary phase cells; resistance to ethanol; spore development; thermoresistance during sporulation, spore germination, thermoresistance of mature or germinating spores or survival after long-term storage in stationary phase or as spores [3, 4]. Transcription of the HSP26 gene appears to be regulated by a mechanism of basal repression during growth at normal temperatures and derepression during heat shock [5]. In this work we compare the level of transcription of HSP26 in Saccharomyces cerevisiae YPH499 cells grown in media at optimal (5.0) and low (2.5) pH, acidified with a strong acid (HCl) which exerts its inhibitory effect only by increasing the concentration of protons around the cell, in contrast with organic acids [7]. This yeast strain was previously examined concerning the effect of optimal and low pH on the expression of the plasma membrane H+-ATPase encoding isogenes PMA1 and PMA2[8].

2 Materials and methods

2.1 Yeast strain and growth conditions

S. cerevisiae YPH499 (Mata, ade2-101ochreleu2-D1 his3-D200 ura3-52 trp1-D1 lys2-801amber) [9] cells were batch cultured at 30°C with orbital agitation (150 rpm) in 250 ml Erlenmeyer flasks with 150 ml of liquid medium containing 30 g l−1 glucose, 6.7 g l−1 yeast nitrogen base without amino acids (Difco), 40 mg l−1l-adenine, 20 mg l−1 uracil and a mixture of amino acids [10]. Growth medium pH was adjusted to 2.5 or 5.0 by addition of HCl or NaOH. Growth was monitored by measuring the OD at 600 nm (OD600) after inoculation with cells (initial OD600=0.2±0.02) pregrown until mid-exponential phase (OD600=1.6±0.1) in medium at pH 4.0.

2.2 HSP26 mRNA quantification

For HSP26 mRNA quantification, total RNA was extracted from cells harvested during growth in media at initial pH 2.5 or 5.0 according to the method of Schmitt et al. [11]. Aurintricarboxylic acid (Sigma), an inhibitor of intracellular RNases, was added to cell suspensions at a concentration of 50 mM. Northern blot hybridizations were carried out as previously described [12]. The quantity of total RNA in each sample used for Northern blotting was constant (20 μg (by OD260); this was confirmed by comparing the intensities of methylene-blue-stained rRNA bands on a blotted membrane). The HSP26 specific probe was prepared using BglII-PstI fragment, with 1.2 kb of the HSP26 gene including the coding region [13] and was kindly provided by Dr. P. Bossier and Dr. C. Rodrigues-Pousada (Lab. of Genetics, Gulbenkian Institute of Science, Oeiras, Portugal). The relative intensities of the hybridization signals on autoradiograms were quantified by densitometry (UVP gel documentation system GDS 2000). The results in Fig. 1 are representative of three independent Northern experiments using extracts from two independently cultivated cells at the same pH and were obtained in the same hybridization experiment probing all the different RNA extracts.

Figure 1

Levels of HSP26 mRNA in S. cerevisiae YPH499 cells during (a) growth carried out in media at initial pH 5.0 (◻) or pH 2.5 (○). mRNAs were quantified by (b) densitometry of (c) autoradiograms from Northern blot hybridizations of total RNAs (20 μg per lane) probed with the HSP26 gene. Relative intensity values are representative of at least three independent Northern experiments using RNA extracts prepared from cells obtained from two independent cultivations at the same pH.

3 Results and discussion

During yeast exponential growth in medium at the optimal pH 5.0 (Fig. 1a), the mRNA levels from HSP26 gene were undetectable but transcription was strongly induced in cells entering the stationary phase (Fig. 1b,c). This growth-phase-dependent transcription of the HSP26 gene was previously observed by Kurtz et al. [2]. The transcription of HSP26 was also strongly induced in exponential growing cells at the inhibitory pH 2.5 (Fig. 1). This observation is one of the few demonstrations of genes induced by low pH in yeast. At the stationary phase of growth at pH 2.5, HSP26 transcripts reached values close to those quantified in stationary cells that had been grown at the optimal pH 5.0 (Fig. 1). These results are consistent with the suggestion by Susek and Lindquist [5] that a common regulatory mechanism is employed for HSP26 transcription in response to various environmental stresses. This hypothesis was based on results which indicate that the sequences responsible for increased expression during stationary phase were redundant in the HSP26 promoter and could not be separated experimentally from the sequence elements responsible for increased expression upon heat shock [5].

Using identical growth conditions and the same yeast strain, we previously reported that acid stress also changed the pattern of expression of PMA1 and PMA2 genes observed at the optimal pH 5.0 [8]. The level of mRNA from the essential plasma membrane ATPase encoding gene PMA1 was found to decrease, as is expected for the majority of yeast genes [14]. Since the extracts used to quantify HSP26 mRNA were also probed with the PMA1 gene [8], these results were used as control experiments which clearly indicate that the increase of HSP26 mRNA accumulation is specific for HSP26 and not for mRNAs in general. However, the efficiency of the promoter of PMA2 gene, the lowly expressed PMA1 isogene, was found to be moderately increased (8-fold) [8] as observed before with other stress conditions [10, 15, 16]. Although the expression of both PMA2 and HSP26 genes was enhanced in cells growing at pH 2.5, this increase was dramatic for HSP26 and moderate for PMA2 and the increased expression of PMA2 gene was reduced as cells entered the stationary phase of growth [8]. Differently, HSP26 transcription is regulated by both stress and development, being derepressed in cells entering the stationary-phase growth and early after transfer to sporulation medium [2]. In the present work, the environmental conditions leading to strongly induction of HSP26 transcription were extended to exponential growth under acid stress, consistent with the idea that different stress conditions might trigger a general response by creating the same intracellular signal. Abnormal or denatured proteins have been suggested to play such a general role but other intracellular parameters like internal acidification, alterations in cytoskeletal structures or changes in secondary messenger levels have to be taken also into consideration (for a review see [14])


This work was supported by JNICT, FEDER, and STRIDE and PRAXIS XXI Programmes (grants: STRDA/C/BIO/387/92 and PRAXIS/2/2.1/BIO/20/94 and a Ph.D. scholarship (BD/2061/92-IF) to V.C.).


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