The International Journal of Biochemistry & Cell Biology
Polyamine modulon in yeast—Stimulation of COX4 synthesis by spermidine at the level of translation
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.
References (50)
- et al.
Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines
J Biol Chem
(1975) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
Import of proteins into mitochondria, Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria
J Biol Chem
(1982) - et al.
Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli
Biochem Biophys Res Commun
(1975) - et al.
New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin
Biochim Biophys Acta
(1981) - et al.
Enhancement of +1 frameshift by polyamines during translation of polypeptide release factor 2 in Escherichia coli
J Biol Chem
(2006) - et al.
Polyamines: mysterious modulators of cellular functions
Biochem Biophys Res Commun
(2000) - et al.
Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems
Biochem Biophys Res Commun
(1979) - et al.
Identification of the polyamine-induced protein as a periplasmic oligopeptide binding protein
J Biol Chem
(1990) - et al.
Anti-proliferative properties of DL-α-difluoromethyl ornithine in cultured cells. A consequence of the irreversible inhibition of ornithine decarboxylase
Biochem Biophys Res Commun
(1978)
Polyamine-sensitive magnesium transport in Saccharomyces cerevisiae
Biochim Biophys Acta
Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells
Int J Biochem Cell Biol
Spermidine regulation of protein synthesis at the level of initiation complex formation of Met-tRNAi, mRNA and ribosomes
Biochem Biophys Res Commun
Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae
J Biol Chem
Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae
Biochem Biophys Res Commun
Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae
J Biol Chem
Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver
J Biol Chem
Cytosolic chaperonin is up-regulated during cell growth. Preferential expression and binding to tubulin at G1/S transition through early S phase
J Biol Chem
Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase σ28 subunit
J Biol Chem
Polyamines enhance synthesis of the RNA polymerase σ38 subunit by suppression of an amber termination codon in the open reading frame
J Biol Chem
A unifying model for the role of polyamines in bacterial cell growth, the polyamine modulon
J Biol Chem
Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon AUG in OppA mRNA
J Biol Chem
Translational control of regA, a key gene controlling cell differentiation in Volvox carteri
Development
Polyamine deficiency leads to accumulation of reactive oxygen species in a spe2Δ mutant of Saccharomyces cerevisiae
Yeast
A guide to polyamines
Cited by (27)
Evidence for adduction of biologic amines with reactive metabolite of 8-epidiosbulbin E acetate in vitro and in vivo
2022, Toxicology LettersCitation Excerpt :Polyamines (PAs) are aliphatic polycations having more than two amino groups, extensively distributed in almost all living organisms. As a necessary kind of biologic amines, PAs participate in various intracellular biochemical activities including DNA and protein synthesis (Davidson et al., 1996; Imamura et al., 2016; Uemura et al., 2009), stimulation of cell division and differentiation (Kramer et al., 2001; Guy et al., 2012; Weicht et al., 2018), modulation of ion channel (Williams, 1997), regulation of apoptosis (Nitta et al., 2001) and cell-cell communication activity (Johnson and McCormack, 1999). Putrescine (Put), spermidine (Spd), and spermine (Spm) are three kinds of major PAs.
The functional role of polyamines in eukaryotic cells
2019, International Journal of Biochemistry and Cell BiologyCitation Excerpt :Under these conditions, the content of putrescine and spermidine becomes almost negligible, but that of spermine decreased to approximately 50% of control (Terui et al., 2015). Thus far, eight proteins have been identified as proteins encoded by the polyamine modulon in eukaryotes (Imamura et al., 2016; Matsufuji et al., 1995; Nishimura et al., 2009; Terui et al., 2015; Uemura et al., 2009). Those are Cox4 (one of the subunits of cytochrome C) in yeast, Cct2 (T-complex protein, β-subunit), Hnrp L (heterogenous nuclear riboprotein L), Pgam1 (phosphoglycerate mutase 1), eEF1 A (elongation factor 1 A), p16 (a transcription factor), EXT2 (exostosin glycosyltransferase 2), and antizymes.
Essential, deadly, enigmatic: Polyamine metabolism and roles in fungal cells
2019, Fungal Biology ReviewsCitation Excerpt :The polyamine modulon is a group of genes whose translation is induced by polyamines (Uemura et al., 2009). In yeast, spermidine enhances the translation of COX4, encoding one of the subunits of the cytochrome C oxidase involved in ATP production, by stimulating ribosome shunting of the COX4 mRNA stem-loop structures (Uemura et al., 2009) during intermittent scanning of the 5′ UTR by the 40S ribosomal subunit (Babinger et al., 2006; Yueh and Schneider, 2000). Considering the involvement of spermidine in fundamental aspects of protein synthesis, maintaining the spermidine pool is critical to fungal growth and development.
Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell
2015, Journal of Molecular BiologyCitation Excerpt :Moreover, PAs stimulate the interaction between inefficient initiation codons, UUG and GUG, and the fMet-tRNA, which enhance the translation of RNAs such as cya and cra [128, 131, 134]. In eukaryotes, PAs increase translation of CCT2, HNRP1 and PGAM1 in mammary carcinoma F3A cells, and COX4 in yeast by “ribosome shunting” [130, 135]. In this translation mechanism the 40S ribosome bypasses 5′ UTR regions and secondary structures which usually interfere with translation, to reach the start codon [136].
Acetaldehyde-induced cytotoxicity involves induction of spermine oxidase at the transcriptional level
2013, ToxicologyCitation Excerpt :High polyamine levels are found in growing cells such as epithelial cells in the gastrointestinal tract and cancer cells (Gerner and Meyskens, 2004). Polyamines interact with RNA and stimulate protein synthesis required for cell growth (Igarashi and Kashiwagi, 2006; Uemura et al., 2009). Cellular polyamine levels are regulated by biosynthesis, degradation and transport.