Polyamine modulon in yeast—Stimulation of COX4 synthesis by spermidine at the level of translation

https://doi.org/10.1016/j.biocel.2009.08.010Get rights and content

Abstract

We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation in Escherichia coli and mammalian cells be referred to as a “polyamine modulon”. In Saccharomyces cerevisiae, proteins whose synthesis is enhanced by polyamines at the level of translation were searched for using a polyamine-requiring mutant of S. cerevisiae deficient in ornithine decarboxylase (YPH499 Δspe1). Addition of spermidine to the medium recovered the spermidine content and enhanced cell growth of the YPH499 Δspe1 mutant by 3–5-fold. Under these conditions, synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV), was enhanced by polyamines about 2.5-fold at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem–loop structures (hairpin structures) during the scanning of the 5′-untranslated region (5′-UTR) of COX4 mRNA by 40S ribosomal subunit-Met-tRNAi complex.

Introduction

Polyamines (putrescine, spermidine and spermine) are present at millimolar concentrations in prokaryotic and eukaryotic cells and play regulatory roles in cell growth (Cohen, 1998, Igarashi and Kashiwagi, 2000). Since a decrease or increase in polyamine content greatly diminishes cell growth (Igarashi et al., 1979b, Raj et al., 2001), the intracellular levels of polyamines are closely regulated at various steps including synthesis, degradation, uptake and excretion (Igarashi and Kashiwagi, 1999, Pegg, 1988). Polyamines exist mostly as polyamine–RNA complexes and thus affect various steps of translation (Igarashi and Kashiwagi, 2006, Watanabe et al., 1991). In fact, polyamines stimulate the synthesis of some proteins in vitro and in vivo (Atkins et al., 1975, Igarashi et al., 1974, Kashiwagi et al., 1990) and increase the fidelity of translation (Igarashi et al., 1979a, Jelenc and Kurland, 1979). Polyamines also induce the in vivo assembly of 30S ribosomal subunits (Echandi and Algranti, 1975, Igarashi et al., 1979b).

We have extensively studied the proteins in Escherichia coli whose synthesis is enhanced by polyamines using a polyamine-requiring mutant MA261 (Cunningham-Rundles and Maas, 1975), and proposed that a set of genes whose expression is enhanced by polyamines at the level of translation can be classified as a “polyamine modulon” (Igarashi and Kashiwagi, 2006). There are three different mechanisms underlying polyamine stimulation of the translation of various members of the polyamine modulon. First, polyamine stimulation of protein synthesis takes place when a Shine–Dalgarno (SD) sequence in the mRNA is obscure or is distant from the initiation codon AUG. Polyamines cause structural changes in a region of the SD sequence and the initiation codon AUG of the mRNA, facilitating formation of the initiation complex. This is the case for OppA, a periplasmic substrate binding protein of the oligopeptide uptake system (Yoshida et al., 1999), FecI σ factor (σ18), a σ factor for the iron transport operon (Yoshida et al., 2004), Fis, a global regulator of transcription of some growth-related genes, including genes for rRNA and some tRNAs (Yoshida et al., 2004), RpoN (σ54), a σ factor for the nitrogen metabolism genes (Terui et al., 2007), and H-NS, a positive regulator of expression of genes involved in flagellin synthesis and ribosomal protein synthesis (Terui et al., 2007). Second, polyamines enhance translation initiation from the inefficient initiation codon UUG or GUG such as in cya mRNA encoding adenylate cyclase (Yoshida et al., 2001), or cra mRNA encoding a global transcription factor for a large number of genes involved in glycolysis and glyconeogenesis (Terui et al., 2007). Third, polyamines stimulate read-through of the amber codon UAG-dependent Gln-tRNASupE on ribosome-associated rpoS mRNA encoding σ38, a σ factor for genes expressed at the stationary phase (Yoshida et al., 2002), or stimulate a +1 frameshift at 26th UGA codon of prfB mRNA encoding a polypeptide release factor 2 (Higashi et al., 2006).

Similarly, we have recently identified three kinds of proteins (Cct2, Hnrpl and Pgam1) whose synthesis is enhanced by polyamines at the level of translation using mouse mammary carcinoma FM3A cells (Nishimura et al., 2009). This is the first report of polyamine modulon in eukaryotes, but comparison of protein synthesis was performed using control and polyamine-deficient cells treated with an inhibitor of polyamine biosynthesis, α-difluoromethylornithine (Mamont et al., 1978). This time, we looked for polyamine modulon in eukaryotes using a yeast polyamine-requiring mutant, which is essentially negligible in polyamine content. The addition of spermidine to the medium enhanced cell growth of the polyamine-requiring mutant. Under these conditions, polyamine enhanced synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV) at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem–loop structures during the scanning of 5′-UTR of COX4 mRNA by 40S ribosomal subunit-Met-tRNAi complex (Babinger et al., 2006, Yueh and Schneider, 2000), the same mechanism as polyamine stimulation of Cct2 synthesis in mammalian cells (Nishimura et al., 2009).

Section snippets

Yeast strains and culture conditions

Saccharomyces cerevisiae YPH499 (MATa ade2-101 his3-Δ200 leu2-Δ1 lys2-801 trpΔ63 ura3-52) and YPH499 Δspe1 (MATa ade2-101 his3-Δ200 leu2-Δ1 lys2-801 trpΔ63 ura3-52 Δspe1::Leu2) (Uemura et al., 2007) were cultured at 30 °C in Mg2+-limited CSD medium (Maruyama et al., 1994) which contains 50 μM MgSO4 instead of 2 mM, 30 mg/l lysine, 20 mg/l adenine sulfate, uracil, histidine and tryptophan. When YPH499 was cultured, 30 mg/l leucine was added. YPH499 and YPH499 Δspe1 cells were cultured in the presence

Stimulation of cell growth by spermidine in a polyamine-requiring mutant

A polyamine-requiring mutant YPH499 Δspe1, in which putrescine cannot be synthesized, was used to study the effects of polyamines on gene expression at the level of translation. As shown in Fig. 1A, cell growth was enhanced by 0.1 mM spermidine more than 3-fold. Spermidine was used because it is the major polyamine in yeast (Tachihara et al., 2005). Under these conditions, significant amounts of spermidine were accumulated in cells (Fig. 1B). The effects on cell growth following addition of

Discussion

In this study, we looked for proteins in S. cerevisiae whose synthesis is enhanced by polyamines at the level of translation. In E. coli, polyamine stimulation of protein synthesis occurs by three different mechanisms. One mechanism involves a characteristic SD sequence of the mRNA, which is important for the initiation of protein synthesis. When the SD sequence of the mRNA is obscure or distant from the initiation codon AUG, polyamines stimulate synthesis of the cognate protein. In eukaryotes,

Acknowledgements

We thank Dr. K. Williams for his help in preparing the manuscript. We also thank Dr. D.E. Ting for kindly supplying antibody against COX4. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology, Japan.

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