Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae

Abstract

As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S. cerevisiae genome.T he regulation of ornithine decarboxylase has been studied extensively in a variety of cells and has been shown to involve a very unusual regulatory mechanism. In this system, a specific and unusual protein, called antizyme, is synthesized in response to spermidine͞spermine administration. This protein not only inhibits ornithine decarboxylase but also is important in preparing the ornithine decarboxylase protein for degradation by proteasomes without ubiquitination (reviewed in refs. 1-3). A similar protein has been shown recently to be synthesized in Schizosaccharomyces pombe after the administration of spermidine (4, 5). However, no direct evidence for the presence of an antizyme-like homologue has been demonstrated in Saccharomyces cerevisiae, despite numerous attempts by a number of investigators, nor has an antizyme homologue been found in the sequence of the S. cerevisiae genome (6). In view of this difference, we have been interested in extending our studies on the factors involved in the regulation of ornithine decarboxylase by spermidine in S. cerevisiae, and, in particular, on the factors involved in the degradation of the enzyme, especially because in S. cerevisiae, regulation of the rate of degradation of ornithine decarboxylase seems to be more important than regulation of its biosynthesis (7,8). Materials and MethodsSubcloning of the Yeast SPE1 Gene into a Yeast Constitutive Expression Vector. To obtain a yeast strain containing a larger amount of ornithine decarboxylase, the SPE1 gene was inserted into plasmid pVT101U obtained from T. Vernet (9). For this construction, we digested the 2,955-bp fragment from pSPE1U (10) with SstI and HindIII (partial) and inserted the resultant fragment into the multicloning site of plasmid pVT101U. With this plasmid ...

Studies on the regulation of ornithine decarboxylase in yeast: Effect of deletion in the MEU1 gene

“…7). This result is what would be expected from the many previous studies on the regulation of ODC by spermidine in yeast (18,22,28,29) and other systems (23)(24)(25). In murine lymphoblasts deficient in methylthioadenosine phosphorylase (MTAP), spermidine addition also suppressed ODC activity (34).…”

“…Immunoblotting was performed on poly(vinylidene difluoride) (PVDF) membrane (Invitrogen). Immunodetection was carried out by using an antibody (1:5,000 dilution) raised against recombinant yeast ODC protein (18). Immunospecificity of the antibody was enhanced by prior adsorption of the antibody with an spe1⌬ extract that does not contain ODC protein.…”

“…The availability of these yeast mutants deleted in the methylthioadenosine phosphorylase gene permits more extensive studies on the role of this gene and the methionine salvage pathway in polyamine metabolism in yeast, and a comparison of these effects in yeast with those reported in mammalian cells. Therefore, as an extension of our work on the regulation of ornithine decarboxylase and of polyamine pools in yeast (18), we have now studied the effect of the meu1⌬ deletion on these parameters. Our findings were different from those reported by Subhi et al (17) on the effects of this mutation, and data are presented on the probable reasons for some of these differences.…”

Polyamine metabolism during the germination of Sclerotinia sclerotiorum ascospores and its relation with host infection

“…In mammalian cells, ODC levels are regulated by a sophisticated mechanism based on a protein known as antizyme (Matsufuji et al, 1995). Also, a SPD-mediated mechanism dependent on protein synthesis for the regulation of ODC levels has recently been reported to operate in Saccharomyces cerevissiae (Gupta et al, 2001) and Schizosaccharomyces pombe (Chattopadhyay et al, 2001). On the basis of the results presented in this paper, we consider interesting to evaluate the existence of a similar mechanism for the regulation of ODC levels in germinating S. sclerotiorum ascospores, proven that this subject has not been investigated in filamentous fungi so far.…”

BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development

“…Yeast cells were cultured as described by Gupta [36]. After a single clone was cultured in YPD medium for 24 h, the 10-µL cultured cells were added to 5-mL SD medium with or without supplement of spermidine and were incubated overnight at 30 °C.…”