Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine

Abstract

Spermidine and its derivative, hypusinated eIF5A, are essential for the growth of Saccharomyces cerevisiae. Very low concentrations of spermidine (10⁻⁸ M) are sufficient for the growth of S. cerevisiae polyamine auxotrophs (spe1Δ, spe2Δ, and spe3Δ). Under these conditions, even though the growth rate is near normal, the internal concentration of spermidine is <0.2% of the spermidine concentration present in wild-type cells. When spe2Δ cells are grown with low concentrations of spermidine, there is a large decrease in the amount of hypusinated eukaryotic initiation factor 5A (eIF5A) (1/20 of normal), even though there is no change in the amount of total (modified plus unmodified) eIF5A. It is striking that, as intracellular spermidine becomes limiting, an increasing portion of it (up to 54%) is used for the hypusine modification of eIF5A. These data indicate that hypusine modification of eIF5A is a most important function for spermidine in supporting the growth of S. cerevisiae polyamine auxotrophs.

Polyamines (putrescine, spermidine, and spermine) (Scheme 1) are present in micromolar-to-millimolar concentrations in prokaryotic and eukaryotic cells and play important roles in cell growth and development (1, 2). Intracellular levels of polyamines are regulated at various steps, including synthesis, degradation, uptake, and excretion, and cells have developed intricate mechanisms to ensure tight regulation of intracellular polyamine pools (3, 4). We have found that, of the three amines, spermidine is most important for cell growth, and near-normal growth is obtained in mutant cultures containing extremely low concentrations of spermidine (5–7).

Scheme 1.

One of the important functions of spermidine is its role as a substrate for the hypusine modification of the putative eukaryotic translation initiation factor 5A (eIF5A) (Scheme 1). eIF5A is a highly conserved and essential protein present in all organisms from archaebacteria to mammals (8–10), and the hypusine/deoxyhypusine modification of eIF5A is essential in all eukaryotic cells (11). Hypusine was first isolated from a brain extract by Shiba et al. (12), and later Folk and colleagues (13–15) showed that the eIF5A precursor undergoes a unique posttranslational modification to form the hypusine residue. This modification occurs exclusively in this protein and proceeds by two consecutive steps. First, precursor eIF5A is modified by deoxyhypusine synthase, where the aminobutyl group of spermidine is attached to a specific lysine residue, followed by hydroxylation of the intermediate by deoxyhypusine hydroxylase to form the active hypusinated eIF5A (16).

Hypusine modification by spermidine is essential for growth and protein synthesis (8, 10, 11) and in our current studies, we show that when a polyamine auxotroph is grown in the presence of very limiting concentrations of spermidine, most of the spermidine is used for the modification of eIF5A. Further support for the concept that this modification of eIF5A is the major function of spermidine, particularly when grown with limiting concentrations of spermidine is that several of the functions that have previously been reported for spermidine have also been described for hypusinated eIF5A in yeast and mammalian cells (5, 11, 16–20).

Results

Comparison of Intracellular Polyamine Content and Morphology of Wild-Type Cells and Polyamine Auxotrophs Grown on Low Concentrations of Spermidine.

Wild-type Saccharomyces cerevisiae cells contain large amounts of polyamines (estimated internal concentration of ≈0.003 M) (Table 1, Y534; ref. 21). Surprisingly, we found that this high internal concentration of polyamines is not needed for yeast growth; indeed, near-normal growth is found when polyamine auxotrophs are grown in 10⁻⁸ M spermidine and the internal concentration of spermidine is very low (1/240 of wild-type cells) (Table 1, Y535, Y536, and Y537). Data presented in Table 1 also show that the absence of putrescine does not affect the growth rate of a spe1Δ auxotroph (Y535), if the medium contains 10⁻⁸ M spermidine.

Table 1.

Comparison of polyamine content of wild-type cells and of polyamine auxotrophs grown in 10⁻⁸ M spermidine (nmol/mg protein)

Strain	Genotype*	Doubling time, h	Putrescine	Spermidine	Spermine
Y534	Wild type (BY4741)	1.75	1	24	2.3
Y535†	KANMX::spe1Δ (Y5034)	2.1	0	0‡	0.03‡
Y536†	KANMX::spe2Δ (Y1743)	2.2	36	0.1‡	0‡
Y537†	KANMX::spe3Δ (Y5488)	2.2	55	0.1‡	0‡

One milliliter of cell culture at 1.0 OD₆₀₀ contained ≈ mg of wet weight cells (≈200 μg of protein).

*With reference to polyamine biosynthetic genes. Saccharomyces Genome Deletion Project, Stanford University, Stanford, CA; Identification number (Open Biosystem) is shown in parentheses.

^†The medium contained 10⁻⁸ M spermidine.

^‡The limit of detection is 0.03 nmol per mg of protein, and the limit of accuracy for the assay method is 0.05 nmol spermidine or spermine per mg of protein.

As seen in Fig. 1A (for spe2Δ mutant Y536), the growth rate in 10⁻⁸ M spermidine was near normal (2-h doubling time). At this concentration (Fig. 1B, point A), there were no gross morphological changes or changes in the nuclear DNA as observed by DAPI staining (Fig. 1B). When these cultures were further diluted 1:10 into fresh medium without added spermidine (calculated 10⁻⁹ M spermidine in the culture), growth continued at the same rate for ≈2 h but then gradually decreased. No gross morphological changes were seen at 6 h (Fig. 1B, point B), but after 24 h of growth (Fig. 1B, point C) in amine-free medium, the growth rate was very slow and the cells showed the abnormal morphology, enlarged size, reduced budding and diffuse nuclear staining that we described earlier (5, 19) (Fig. 1B). After 60–90 h in the amine-deficient medium, growth stopped, and a large portion of cells was not viable (19).

Fig. 1.

Effect of spermidine deprivation on growth (A), morphological abnormalities (B), and rate of protein synthesis (C) of spe2Δ cells. (B) Cultures were grown in 10⁻⁸ M spermidine and diluted at zero time to an OD₆₀₀ of 0.001. The cultures were grown to an OD₆₀₀ of 1 and at time A diluted into amine-free medium. The optical density values have been corrected for these dilutions. At times A, B, and C, cultures were taken for morphological studies. Cultures were harvested, fixed, and stained with DAPI and observed with a DAPI filter set (Lower). The same cells were also observed by DIC microscopy (Upper). (Scale bar = 10 μm.) Parallel cultures were grown at higher spermidine concentrations as a control. In a separate experiment, to determine the protein synthesis rate, ³[H]-leucine was added into spe2Δ cultures grown in different spermidine concentrations, and to wild-type yeast cells. The cultures were incubated for 20 min, harvested, washed, and ³[H]-leucine incorporation into protein was determined in TCA precipitates as described in Materials and Methods.

Because the spe2Δ cells accumulated large amounts of putrescine that might be responsible for the growth and morphological defects, we carried out comparable studies on a spe1Δ spe2Δ mutant that contained no putrescine or other polyamines and found the same results as described above for the spe2Δ mutant (data not shown).

We wished to examine how depletion of cellular polyamine levels and concomitant decrease in hypusinated eIF5A (see below) affects protein synthesis in vivo by administering a pulse of ³[H]-leucine to spe2Δ cells (Y536) and to wild-type cells (Y534). The results (Fig. 1C) show there is only a small decrease in protein synthesis in cells grown in 10⁻⁸ M spermidine compared with wild type, but a 70% decrease in protein synthesis in cells grown in 10⁻⁹ M spermidine. Thus, the protein synthesis data are consistent with the growth-rate data (Fig. 1A).

Spermidine and Hypusine Content of a spe2Δ Auxotroph Grown in Different Concentrations of [³H]-Spermidine.

We grew the spe2Δ cells in medium containing [³H]-spermidine to obtain accurate quantitation of the intracellular spermidine and hypusine content of cells grown on low spermidine concentrations.

spe2Δ cells were grown in different concentrations of [³H]-spermidine and harvested when the OD₆₀₀ nm was 1.0. At each concentration, >85% of the added radioactivity was taken up by the cells. The cells were extracted with trichloroacetic acid (TCA), and the extract was subjected to HPLC as described in Materials and Methods. Most of the radioactivity (>90%) was found in the spermidine and diaminopropane fractions. The spermidine area was collected, and the radioactivity [disintegrations per minute (DPM)] in this fraction was measured. The concentration of spermidine in nanomoles was calculated by dividing the radioactivity in this fraction by the specific activity of the commercial spermidine (3.2 × 10⁷ DPM per nmol) added to the culture, because in the spe2Δ mutant, no endogenous cold spermidine is synthesized. Based on these assays, the internal spermidine concentration was estimated as 0.36 mM in spe2Δ cells grown in 10⁻⁶ M spermidine, 0.027 mM in cells grown in 10⁻⁷ M spermidine, 0.002 mM in cells grown in 10⁻⁸ M spermidine, and 0.0002 mM in cells grown in 10⁻⁹ M spermidine, whereas that for the wild-type cells was estimated to be ≈2 mM (see below).

For each concentration, a portion of intracellular radioactivity was found in the TCA-insoluble fraction. After thorough washing of the TCA precipitate, the proteins were hydrolyzed in HCl, and the protein-bound radioactive component (>95%) was identified as hypusine by ion exchange chromatography. The total amount of hypusinated eIF5A was calculated for each culture by dividing the total radioactivity in the TCA-insoluble fraction by the specific activity of the aminobutyl moiety (1.6 × 10⁷ DPM/nmol) of [1,8-³H]-spermidine added to the culture. As shown in Table 2, the amount of hypusinated eIF5A markedly decreases when cells are grown in lower concentrations of spermidine.

Table 2.

Labeling of yeast cells with [³H]-spermidine

Cultures per treatment	Spermidine*		Hypusine*		Hypusine/(spermidine+hypusine), %
	DPM/ml cells	nmol/mg protein	DPM/ml cells	nmol/mg protein
Wild type (Y534) (with 5 μCi/ml [3H]-spermidine)	4.86 × 106	20	3.46 × 104	0.28	1.4
Y536-10−6 M [3H]-spermidine	2.70 × 107	4.2	3.15 × 105	0.098	2.3
Y536-10−7 M [3H]-spermidine	1.84 × 106	0.29	1.60 × 105	0.05	15
Y536-10−8 M [3H]-spermidine	1.39 × 105	0.022	4.13 × 104	0.013	37
Y536-10−9 M [3H]-spermidine	1.10 × 104	0.0017	6.35 × 103	0.002	54

Spermidine and hypusine content of wild-type and spe2Δ cultures grown in different concentrations of [³H]-spermidine.

*The spermidine and hypusine content in the wild-type and spe2Δ strains was estimated after chromatographic separation, as described in the text.

A comparable experiment was also carried out with wild-type cells by growing the cells with ≈3.0 × 10⁻⁸ M [³H]-spermidine. The spemidine content of wild-type cells was 20 nmol/mg protein (Table 2), which is equivalent to 2 mM intracellular concentration. To obtain the specific activity of the spermidine in the cells, we chromatographed the TCA extract on an HPLC column; >90% of the counts migrated with the spermidine peak, whereas a small portion (<10%) was found in the diaminopropane and spermine areas. The amount of spermidine in the spermidine peak was determined by the o-phthalaldehyde reaction; the radioactivity was measured and the specific activity calculated (this calculation is an approximation, because the specific activity of the spermidine isolated from the cells at the end of the incubation was lower than that present earlier in the incubation because of the synthesis of cold spermidine during growth).

To obtain the number of nanomoles of hypusine present in the TCA-insoluble fraction of the wild-type cells, the number of counts in this fraction was divided by the specific activity of the aminobutyl moiety of the spermidine isolated from the cells (see above). The amount of modified eIF5A present in wild-type cells was estimated to be 0.28 nmol/mg protein in the wild-type (Y534) cells. Strikingly, the cells grown in 10⁻⁸ M spermidine contained only ≈5% of the hypusinated eIF5A content of wild-type cells or 13% of that of spe2Δ cells grown in 10⁻⁶ M spermidine. The spe2Δ culture grown in 10⁻⁹ M spermidine contained <2% of the hypusinated eIF5A found in spe2Δ cells grown in 10⁻⁶ M spermidine. The finding that growth rates and protein synthesis are only slightly decreased in 10⁻⁸ M spermidine, even though the cellular spermidine and hypusine are reduced by ≈1,000- and ≈20-fold, respectively, indicates that normal levels of both spermidine and hypusine are in large excess over that needed for growth.

Interestingly, the percentage of the radioactivity in the protein-bound hypusine was remarkably higher in the cells grown in the lower concentration of spermidine (Table 2). Thus, in spe2Δ cells grown in 10⁻⁸ M spermidine, 37% of the intracellular spermidine was converted to hypusine and in the cells grown in 10⁻⁹ M spermidine, 54% of the radioactive spermidine was converted to hypusine. This is in contrast to wild-type cells or spe2Δ cells grown in 10⁻⁶ M spermidine, where only 1.4% or 2.3% of the spermidine was used for hypusine synthesis. That an increasing percentage of spermidine is mobilized for hypusine synthesis as spermidine becomes limiting suggests the most important function of cellular spermidine in yeast is the hypusine modification of eIF5A.

We examined the radio-labeled protein by fluorography after SDS/PAGE (Fig. 2A). In all cases, the radioactivity in the TCA-insoluble fraction migrated as a single band corresponding to yeast eIF5A. The intensities of the labeled eIF5A band in the fluorogram decreased in cells cultured in lower concentration of [³H]-spermidine and are consistent with the amounts of hypusine (Table 2) estimated from acid hydrolysates of TCA precipitated proteins. We also measured the amount of total eIF5A in these cells by Western blot analysis (Fig. 2B) using a rabbit polyclonal antibody against recombinant yeast eIF5Aa (Tif51a), which recognizes the unmodified eIF5A precursor as well as the hypusinated eIF5A. In contrast to the fluorogram (Fig. 2A), which showed a sharp decline in the hypusinated eIF5A content in spe2Δ cells cultured in lower spermidine concentrations, the total eIF5A protein (unmodified plus modified) did not change in cultures grown with different concentration of spermidine (Fig. 2B), an indication that the unmodified eIF5A precursor accumulates in spermidine-deprived spe2Δ cells. Thus spermidine depletion does not seem to inhibit de novo synthesis of eIF5A protein but limits only its hypusine modification.

Fig. 2.

Level of hypusinated eIF5A (A) and total eIF5A (B) in wild-type and different spermidine-supplemented spe2Δ cultures. The yeast spe2Δ culture was grown in 10⁻⁸ M spermidine and then diluted to an OD₆₀₀ of 0.001 in media containing 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ M [³H]-spermidine, as indicated. The wild-type culture was also grown in the presence of 5 μCi/ml [³H]-spermidine. Cultures were harvested at an OD₆₀₀ of 1 and subjected to analysis by SDS/PAGE. The migration of [³H]-spermidine incorporated as hypusine in eIF5A, and standard yeast eIF5A were determined by fluorography and staining. Note that the [³H]-spermidine level in wild-type cells is diluted by intracellular biosynthesis of cold spermidine (A). Total eIF5A (hypusinated and precursor) was determined in the above cultures by Western blot analysis against rabbit antiyeast eIF5A antibody, kindly provided by Sandro Valentini (São Paulo State University, Universidade Estadual Paulista, Araraquara, SP, Brazil) (B). Equal loading of proteins in the gel lanes in A and B was confirmed by staining with Coomassie and ponceau-S, respectively [see supporting information (SI) Fig. 4].

Growth Effects of Spermidine Analogs in Complete Absence of Spermidine.

A number of polyamine analogs were also tested (at 10⁻⁷ M concentrations) for their ability to support growth of the spe2Δ auxotroph in the absence of added spermidine. Only 1-methylspermidine supported growth of the S. cerevisiae polyamine auxotroph (2.3-h as compared with 1.7-h doubling time for spermidine). Furthermore, the S-stereoisomer of 1-methylspermidine showed better growth than the R-isomer (2.1 h for the S-isomer as compared with 2.9 h for the R-isomer). Addition of 8-methylspermidine, caldine, and putrescine had no effect on growth.

Discussion

The data in this paper, plus our previous reports (5–7), demonstrate that very low concentrations of spermidine (10⁻⁸ M) are sufficient for near-normal growth and protein synthesis of a spe2Δ polyamine auxotroph of S. cerevisiae. In view of the importance of spermidine as a precursor of hypusine (in eIF5A), we were interested in what effect, if any, this large decrease in the internal concentration of spermidine in polyamine auxotroph cells (grown in 10⁻⁸ M spermidine) might have on the concentration of total eIF5A (i.e., modified and unmodified) and on the concentration of modified (hypusinated) eIF5A. As shown in Fig. 2B, we found that the total amount of eIF5A was essentially unchanged, but that there was a large decrease (1/20) in the amount of hypusinated eIF5A (Table 2 and Fig. 2A). Because the polyamine auxotrophs grow at near-normal growth rates in 10⁻⁸ M spermidine, these data were surprising in indicating that normally the yeast cells contain a many-fold excess of hypusinated eIF5A than is needed for optimum growth. These observations are important in emphasizing the need for marked depletion of the hypusinated eIF5A in any experiments designed to test the biologic effect of this modification and may account for the difficulties observed in the past experiments designed to evaluate the importance of eIF5A on protein synthesis (11).

Most interesting was the observation (Table 2) that in the spe2Δ cells grown on the lower concentrations of spermidine, a much larger percentage of the available spermidine was used for the synthesis of hypusine; i.e., 54% of the available spermidine was used for hypusine biosynthesis in cells grown in very limiting spermidine (10⁻⁹ M) vs. 1–3% for cells grown in 10⁻⁶ M spermidine or in a prototrophic strain. Considering the known binding of spermidine to other acidic macromolecules in cells, it is remarkable that 54% of cellular spermidine can be used for hypusine synthesis under these limiting conditions. One explanation for these results could be that the hypusine modification is an enzymatic irreversible reaction, and thus may act as a “sink” for any available spermidine. Our results are consistent with the observation of Gerner et al. (22) that when spermidine was added to α-difluoromethylornithine (DFMO)-treated rat hepatoma cells, it was immediately incorporated into eIF5A before the intracellular content of free spermidine was elevated.

When the cells are incubated in an even lower concentration of spermidine (10⁻⁹ M or lower), the intracellular spermidine and modified eIF5A concentrations go below the critical levels, and growth gradually stops. The importance of hypusine modification for cell growth is further substantiated by finding that, of the various analogs tested, only 1-methylspermidine supported growth of the S. cerevisiae polyamine auxotroph, and this analog is the one that has been definitively shown in in vitro studies to act as a substrate for the modification of eIF5A (16, 23). Also, of the R- and S-stereoisomers of 1-methylspermidine, the S-isomer was better in supporting growth, and this isomer had been reported to be a better substrate for modification of eIF5A in mammalian cells (24). Previously, we have shown that in a polyamine auxotroph that cannot convert spermine to spermidine because of a deletion in the FMS1 gene (spermine oxidase), spermine does not support growth (7), and it has been shown that spermine cannot be used as a substrate for deoxyhypusine synthase (16). Further support for the major importance of hypusine formation in spermidine-limited cultures is that the morphological defects we observed previously and in our current studies (Fig. 1B) during polyamine deprivation are similar in eIF5A-depleted cells (containing a normal amount of polyamines). For example, either eIF5A or spermidine depletion results in G₁-S arrest in the cell cycle of yeast and mammalian cells (11, 16, 18); abnormalities in actin polarization have been reported because of deprivation of either spermidine or eIF5A (5, 18, 20, 25). Recent studies from our laboratory (19) and by Schrader et al. (26) have reported apoptotic cell death in yeast during spermidine depletion (19) or in eIF5A temperature sensitive mutants (26). Like eIF5A mutants, spermidine-depleted yeast cells are highly sensitive to protein synthesis inhibitors, such as paromomycin (27, 28). Taken together, these findings strongly suggest that hypusine modification of eIF5A is a most critical function for spermidine in supporting the growth of S. cerevisiae. These findings are also important in indicating the need for reinvestigating the concentrations of spermidine required for the many reported functions for polyamines and for evaluating whether they are primary effects of the polyamines or secondary to the growth defects resulting from inadequate eIF5A modification.

The function of polyamines in mammalian cells may be much more complex, in view of their implicated roles in the regulation of diverse cellular activities such as transcriptional, translational, and posttranslational levels affecting proliferation, transformation, differentiation, and apoptosis. Indeed, Nishimura et al. (29), using inhibitors, have demonstrated an independent role for polyamines and eIF5A in supporting the proliferation of mouse mammary carcinoma cells. In addition, Nagarajan et al. (30) have shown that either spermidine or spermine derivatives can support growth of polyamine depleted SV-3T3 cells without the need for metabolic conversion. Furthermore, cytostasis of mammalian cells induced by the ornithine decarboxylase (ODC) inhibitor DFMO (24) or a S-adenosylmethionine decarboxylase inhibitor (25) has been reported to have two phases, an acute initial phase due to reduction of total cellular polyamines and a delayed second phase due to eventual deprivation of eIF5A.

Because the polyamine auxotrophs are able to grow at near-normal rates in media containing 10⁻⁸ M spermidine even with a low internal concentration of spermidine and of eIF5A, these findings lead to the unanswered question of why wild-type cells have a large excess of internal spermidine (1,000-fold more than needed for near-optimum growth) and of hypusinated eIF5A (≈20-fold more than needed for growth). It is also noteworthy that prokaryotes such as Escherichia coli do not have hypusine or eIF5A and yet contain large amounts of putrescine and spermidine that appear to have a number of physiological effects such as transcription, translation, and protection from stress (4, 31–34). It is also surprising that in vivo protein synthesis (Fig. 1B) was nearly as efficient in cultures grown in 10⁻⁸ M spermidine as in wild-type cells, in view of the reported effects of spermidine and polyamines on the stabilization of nucleic acids and on the efficiency and fidelity of in vitro translation. Further studies are needed to test whether the excess amounts of cellular polyamines in yeast and bacteria may be important for the fidelity of replication, transcription, and translation and for the long-term genetic preservation and survival of the organism.

Materials and Methods

Strains and Growth Conditions.

The strains used in this study are listed in Table 1 (BY4741 background, Mat a his3 leu2 met15 ura3) and were maintained on yeast extract peptone adenine dextrose (YPAD) agar plates. Before use, the strains were partially deprived of endogenous amines by incubation in minimal medium containing 10⁻⁸ M spermidine. The minimal medium consisted of 0.67% yeast nitrogen base (Q-BIOgene), 2% glucose, and any required supplements; to each liter of medium, 10 ml of 1 M K₂HPO₄ was added, resulting in an initial pH of 6.5. All incubations were carried out at 30°C with shaking in air. The cells were diluted 1:1,000 into the same medium (containing 10⁻⁸ M spermidine) to give a calculated optical density at zero time of ≈0.001. The cultures were then incubated and harvested by centrifugation when the OD₆₀₀ reached 1.

Polyamine Analysis by HPLC.

The cell pellet was extracted with 5 volumes of 10% perchloric acid or 10% trichloroacetic acid. Aliquots (usually 50 μl) were assayed for polyamines by HPLC chromatography essentially as described by Murakami and Hayashi (35) using a Shim-pack column (Shimadzu, catalogue no. ISC-05/S0504). Postcolumn fluorometric determination of polyamines was performed by reaction with o-phthalaldehyde (6), and data were collected by using Powerchrome software (eDAQ). Intracellular polyamine concentrations were calculated by assuming that 90% of the yeast cells wet weight is water.

Determination of Intracellular Spermidine and Hypusine Content of a Wild-Type and a spe2Δ Mutant Cultured in the Presence of [³H]-Spermidine.

The yeast cells were cultured in 10 ml of minimal medium containing the indicated amounts of [1,8-³H]-spermidine (16.6 Ci/mmol, Perkin–Elmer/NEN, catalogue no. NET522; HPLC analysis showed >90% of the counts migrated in the spermidine area) overnight. Cells (at 1.0–1.6 OD₆₀₀) were harvested and washed with fresh culture medium. To each cell pellet, 0.2 ml of 10% TCA was added and kept on ice for 20 min. After centrifugation (14,000 × g) for 5 min, the TCA supernatant was separated for polyamine and radioactivity analyses. The TCA precipitates were resuspended in 1 ml of 10% TCA containing 1 mM each unlabeled polyamines (putrescine, spermidine, spermine) to remove any radioactive spermidine noncovalently bound to the TCA precipitates. This wash was repeated (>3×) until there was no radioactivity in the TCA wash. A portion of the washed TCA precipitate (1/10 of the total) was resuspended in 0.1 ml of 0.2 M NaOH, and an aliquot was used for protein determination by the bicinchoninic acid protein assay (Promega). Another portion of the precipitate (1/5 of the total) was dissolved in the SDS sample buffer, subjected to SDS/PAGE, and used for fluorographic detection of [³H]-labeled hypusine-containing protein, eIF5A and for detection of total eIF5A protein (unmodified precursor and modified eIF5A) by Western blotting (11). A third portion of the washed TCA precipitate (1/5) was hydrolyzed in 6 M HCl at 108°C overnight, and the amount of radioactive hypusine present was determined after ion exchange chromatography as described, with slight modification (13).

Assay of Protein Synthesis in Wild-Type and spe2Δ Mutant Grown in Different Spermidine Concentrations.

The wild-type (Y534) and mutant strains (Y536) were cultured in minimal medium containing the indicated concentrations of spermidine. Duplicates of 3.75 ml of the exponentially growing cells (OD₆₀₀ nm of 0.3–0.4) were harvested by centrifugation at 2,000 × g for 5 min at room temperature, and 2.75 ml of medium was removed leaving 1 ml of medium in which cells were resuspended. 30 μCi of [³H]-leucine [l-[4,5-³H(N)]leucine, 60 Ci/mmol, Perkin–Elmer/NEN catalogue no. NET135H] was added, and cells were incubated for 20 min at 30°C with shaking. The reaction was stopped by addition of 0.5 ml of ice-cold stop solution (1 mg/ml of cycloheximide and 3 mg/ml of leucine). Cells were kept on ice for 1 min and centrifuged in the microfuge. The cell pellets were resuspended in 1 ml of 15% TCA, and the mixture was heated at 95°C for 10 min. The samples were cooled on ice and after centrifugation, the TCA precipitate was washed three times with 10% TCA to remove free [³H]-leucine. A portion of the washed TCA precipitate (1/5) was hydrolyzed in 6 M HCl at 108°C overnight, and the radioactivity in the acid hydrolysate was counted to determine the amount of [³H]-leucine incorporated into proteins. Another portion (1/5) of the TCA precipitate was resuspended in 0.1 ml of 0.2 M NaOH, and an aliquot was used for protein determination by the BCA method.

Microscopic Studies.

For nuclear staining, cells were grown as above in different concentrations of spermidine, harvested, and fixed with 70% ethanol for 1 h on ice, and the cells were resuspended in PBS containing 1 μg/ml DAPI (Sigma) and 1 mg/ml p-phenylenediamine (Sigma) for 30 min. The cells were then washed twice in PBS and observed in a fluorescence microscope (Zeiss Axiophot) equipped with a digital camera (Cool Snap HQ, Photometrics), and IP Lab software (for capture) through a DAPI filter set. The cells of the same fields were also visualized by differential interference contrast (Nomarski) microscopy.