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Original Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2006, 38: 293-298
doi:10.1111/j.1745-7270.2006.00162.x

partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

Yong-Fu LI¹ and Feng SHI²*

1 School of Food Science and Technology, and

2 The Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China

Received: January 9, 2006

Accepted: February 22, 2006

*Corresponding author: Tel, 86-510-85864741; Fax, 86-510-85806493; E-mail, [email protected]

Abstract������� Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

Key words������� NAD kinase; Saccharomyces cerevisiae; mitochondrial function; temperature sensitivity; subcellular localization

In a model eukaryotic organism, the yeast Saccharomyces cerevisiae, three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, were found and all identified as ATP-NADH kinases [1-3]. The localizations of Yef1p and Utr1p were predicted by computer program analysis using iPSORT (http://hc.ims.u-tokyo.ac.jp/iPSORT) for the detection of N-terminal Protein SORTing signals and prediction protein localization sites [4], which did not show any positive targeting or signal sequences, implying they were probably cytosolic enzymes, whereas Pos5p was confirmed to localize in the mitochondrial matrix [2,3]. Pos5p plays a significant role in mitochondrial NADPH biosynthesis and has been shown to be important to several NADPH-requisite processes of mitochondria, for example, resistance to oxidative stress, arginine biosynthesis, respiration, mitochondrial iron homeostasis and mitochondrial DNA stability [2,3]. The pos5 cells showed growth defects in the presence of oxidative damage, in the presence of glycerol as the sole carbon source and in a medium without arginine [1,2]. They also accumulated high mitochondrial iron and defected in the mitochondrial Fe-S cluster-containing enzymes [2]. Furthermore, frame-shift mutations in mitochondrial DNA were also increased [3].

Compared with the pos5 single mutant, pos5 double mutants (yef1pos5 and utr1pos5) showed greater mitochondrial dysfunction and temperature sensitivity. The temperature sensitivity of the triple mutant (utr1yef1pos5) was even more extreme, indicating the partial contribution of YEF1 and UTR1 gene products to Pos5p function only in the absence of Pos5p [1]. In order to confirm this, we constructed high-copy vectors carrying the YEF1 or UTR1 gene, in which the green fluorescent protein (GFP) gene was inserted into the 3' terminus, then introduced them into pos5 single, double and triple mutants (pos5, utr1pos5, yef1pos5 and utr1yef1pos5). Through checking the growth phenotypes of these positive transformants, we wanted to clarify the partial rescue of pos5 mutants by YEF1 and UTR1 genes and their potential supplying ability for mitochondrial NADP and/or NADPH. We also attempted to examine the localization of Yef1p and Utr1p in yeast cells using the label of the GFP reporter tag.

Materials and Methods

Strains and media

The yeast strains used in this study are listed in Table 1. Strains of S. cerevisiae were cultured in synthetic dextrose (SD) medium (0.67% yeast nitrogen base without amino acid, 2% glucose and appropriate amino acids; pH 5.0). Glucose was replaced with 3% glycerol in the synthetic glycerol (SG) medium. In order to prepare solid media, liquid media were solidified using 2% agar. To check the growth on solid media, the cells were cultured to saturation at 30 �C, collected, washed three times in sterilized water and diluted in water to A600 of 2.0, 0.2 and 0.02. The diluted cell suspensions (5 ml) were spotted on appropriate solid media, which were then incubated at 30 �C or 37 �C. After 5 d for SD media or 14 d for SG media, photographs were taken.

Construction of YEF1 and UTR1 gene reporter strains

The primers used in this study are listed in Table 2. The YEF1 and UTR1 gene reporter strains were constructed as described elsewhere [5]. GFP-HIS3, flanking approximately 40 nucleotides at the 3' terminal and downstream of YEF1, was amplified by polymerase chain reaction (PCR) with the plasmid pFA6a-GFP(F64A, S65T, R80Q, V163A)-His3MX6 [6] using primers yef1GFPf and yef1hisr, and was introduced into BY4742 (EUROSCARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/) by the lithium acetate method [7] to insert GFP-HIS3 into the 3' terminal of YEF1, resulting in the YEF1 gene reporter strain (yef1::YEF1-GFP-HIS3). Similarly, the GFP-HIS3 flanking the 3' terminal and downstream of UTR1 was obtained using primers utr1GFPf and utr1GFPr, and introduced into BY4742, yielding the UTR1 gene reporter strain (utr1::UTR1-GFP-HIS3). The GFP reporter tag of the YEF1 and UTR1 genes was confirmed by colony PCR using primers yef1upf, pfahis3r, pfahis3f, yef1dnr and utr1up0.5k, pfahis3r, pfahis3f, utr1dnr, respectively.

Construction of YEF1-GFP and UTR1-GFP high-copy plasmids

YEF1-GFP and UTR1-GFP high-copy plasmids were constructed as follows: YEF1-GFP and UTR1-GFP flanking 503 bp upstream of the YEF1 and UTR1 genes were amplified by PCR from genomic DNA of yef1::YEF1-GFP-HIS3 and utr1::UTR1-GFP-HIS3 using the primers yef1up0.5kb, GFPdnr and utr1up0.5kb, GFPdnr, and inserted into the SmaI site of YEplac195 (GenBank accession No. X75459, ATCC87589; S. cerevisiae/Escherichia coli shuttling vector, 2 micron, URA3, lacZ MCS, Apr) [8] to produce YEF1GFP-YEp and UTR1GFP-YEp, respectively.

Construction of YEF1-GFP and UTR1-GFP high-copy gene reporter strains

S. cerevisiae pos5 single, double and triple mutants (pos5, utr1pos5, yef1pos5 and utr1yef1pos5) [1] were transformed with YEF1GFP-YEp, UTR1GFP-YEp and YEplac195, yielding each kind of YEF1, UTR1 high-copy gene reporter strains and negative control strains carrying vector only. Similarly, S. cerevisiae BY4742 was transformed with YEplac195, resulting in positive control strain WT YEplac195.

Microscopic imaging of GFP-tagged strains

Aliquot strains grown to mid-logarithmic phase in SD medium lacking histidine were collected, washed and suspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to A600 of 100. Three microliters of suspensions was analyzed in microscope slides by multiple wavelength fluorescence and visible light microscopy with a digital imaging-capable Olympus (Tokyo, Japan) inverted microscope using an oil-immersed objective at magnification 100�. The differential interference contrast and green fluorescence images were captured at 0.2 s and 10 s after normal and green fluorescence light was emitted, respectively.

Results

Partial rescue of pos5 single mutant by YEF1 and UTR1 genes

According to previous studies, the pos5 single mutant showed temperature sensitivity [1] and mitochondrial dysfunction, such as poor ability for glycerol usage and arginine biosynthesis [1-3]. In order to confirm the contribution of high-copy YEF1 and UTR1 genes to pos5 cells, the growth of pos5 YEF1GFP-YEp, pos5 UTR1GFP-YEp, negative control cells pos5 YEplac195 and positive control cells WT YEplac195 were examined on SD or SG media with or without arginine at 30 �C and on SD medium with arginine at 37 �C (Fig. 1).

Both pos5 YEF1GFP-YEp and pos5 UTR1GFP-YEp cells were able to grow on SG media, whereas pos5 YEplac195 cells were only just able to grow. On SD media with arginine at 37 �C, these two strains grew a little stronger than pos5 YEplac195 cells. But on SD media without arginine, only pos5 YEF1GFP-YEp cells showed a stronger growth than the negative control cells. The results showed that YEF1-GFP in a high-copy vector could express well and partially rescue the temperature sensitivity and mitochondrial dysfunction of pos5 cells, and UTR1-GFP could partially rescue the temperature sensitivity and glycerol growth defect of pos5 cells, suggesting a better contribution of Yef1p and Utr1p, respectively, to Pos5p function.

Partial rescue of pos5 double mutants by YEF1 and UTR1 genes

As the utr1pos5 double mutant showed more acute temperature sensitivity and mitochondrial dysfunction than the pos5 single mutant [1], the growth phenotypes of utr1pos5 YEF1GFP-YEp, utr1pos5 UTR1GFP-YEp, negative control cells utr1pos5 YEplac195 and positive control cells WT YEplac195 were then checked (Fig. 2).

utr1pos5 YEF1GFP-YEp cells grew stronger than utr1pos5 YEplac195 cells, but weaker than WT YEplac195 cells, on all media checked except the control medium, indicating that YEF1-GFP in a high-copy vector could partially rescue the growth defect of utr1pos5 cells, and suggesting the contribution ability of Yef1p to Pos5p functions. The utr1pos5 UTR1GFP-YEp cells grew obviously stronger than the negative control cells only on SG medium with arginine, whereas they grew only slightly stronger on SD medium at 37 �C and on SG medium without arginine. These results indicated that UTR1-GFP in a high-copy vector could partially rescue the glycerol growth defect of pos5 cells, suggesting a weaker contribution of Utr1p to Pos5p functions.

As the yef1pos5 double mutant showed similar temperature sensitivity and mitochondrial dysfunction to that of utr1pos5 cells [1], the growth of yef1pos5 YEF1GFP-YEp, yef1pos5 UTR1GFP-YEp, yef1pos5 YEplac195 and WT YEplac195 on SD media with or without arginine at 30 �C and with arginine at 37 �C were also checked (Fig. 3). Their growth on SG medium was not examined. fig. 3 shows that Yef1p expressed in yef1pos5 YEF1GFP-YEp cells could contribute to Pos5p function of arginine biosynthesis and temperature endurance, whereas Utr1p could only contribute slightly to Pos5p temperature endurance.

Partial rescue of pos5 triple mutant by YEF1 and UTR1 genes

As the utr1yef1pos5 triple mutant showed even more significant temperature sensitivity than pos5 single and double mutants, but not mitochondrial dysfunction [1], the growth of utr1yef1pos5 YEF1GFP-YEp, utr1yef1pos5 UTR1GFP-YEp, negative control cells utr1yef1pos5 YEplac195 and positive control cells WT YEplac195 at 30 �C and 37 �C was checked (Fig. 4), which showed that utr1yef1pos5 YEF1GFP-YEp and utr1yef1pos5 UTR1GFP-YEp grew a little weaker than the positive control cells, but stronger than the negative control cells, indicating the sufficient expression of YEF1-GFP and UTR1-GFP in high-copy vector and the partial rescuing ability for temperature sensitivity of utr1yef1pos5 cells.

The localization of a particular gene product can be observed using the GFP reporter tag in intact cells [9]. In order to determine the subcellular localization of Yef1p and Utr1p in yeast cells, well-expressed utr1yef1pos5 YEF1GFP-YEp and utr1yef1pos5 UTR1GFP-YEp cells growing at 37 �C were selected and observed by fluorescence and visible light microscopy, using pos5::POS5-GFP-HIS3 cells purchased from Invitrogen (Carlsbad, USA) as a control [10,11]. The green fluorescence could not be found in utr1yef1pos5 YEF1GFP-YEp and utr1yef1pos5 UTR1GFP-YEp cells, but could be found in the mitochondria of pos5::POS5-GFP-HIS3 cell (data not shown).

Discussion

Our previous phenotypic study on the single, double and triple mutants for UTR1, YEF1 and POS5 indicated the critical contribution of Pos5p to mitochondrial function and temperature sensitivity, and the partial contribution of Yef1p and Utr1p only in the absence of Pos5p [1]. Here the phenotypic study of pos5 cells carrying high-copy GFP-tagged YEF1 and UTR1 plasmids was performed to confirm such effects of Yef1p and Utr1p on pos5 cells, and to search the well expressed strains to determine the localization of GFP-fused Yef1p and Utr1p.

The phenotypic analysis (Figs. 1-4) reflected the non-specific pleiotropic consequences of Yef1p and Utr1p on cellular physiology, emphasized here as the partial rescue of mitochondrial dysfunction in the absence of Pos5p. The temperature sensitivity and mitochondrial dysfunction of pos5 mutants could be rescued partially by the high-copy plasmid YEF1GFP-YEp, and the temperature sensitivity and glycerol growth defect of pos5 mutants be rescued partially by UTR1GFP-YEp plasmid, so YEF1-GFP and UTR1-GFP genes in these plasmids should be well expressed. Thus the localization of Yef1p and Utr1p could be checked by these high-copy GFP tag systems rather than by genomic one-copy GFP tags of yef1::YEF1-GFP-HIS3 and utr1::UTR1-GFP-HIS3 cells (data not shown) [1]. Unfortunately, the green fluorescence could not be found by fluorescence microscopy, so the subcellular localization of Yef1p and Utr1p in yeast cells could not be determined, indicating the lower expression level of these two NAD kinase homologs, even in these well expressed high-copy systems.

Even with the lower expressed products of the YEF1-GFP and UTR1-GFP genes, the dysfunction of pos5 mutants could be partially compensated, implying that Yef1p and Utr1p could supply part of mitochondrial NADP and/or NADPH in the absence of Pos5p. As Yef1p and Utr1p are presumably cytosolic enzymes, it could be supposed that high amounts of cytosolic NADP or NADPH could be transported weakly into mitochondria through a novel transporter in the mitochondrial inner membrane. The proposed flows of pyridine nucleotides are shown in Fig. 5.

As cytosolic NADPH is mainly supplied by Zwf1p and is needed for methionine biosynthesis, whereas mitochondrial NADPH is mainly supplied by Pos5p and is needed for arginine biosynthesis [2], the methionine auxotrophy of zwf1 and arginine auxotrophy of pos5 cells [1,2] thus indicate (a) the separate supplying of NADPH in cytosol and mitochondria, and (b) Yef1p and Utr1p functionally operate as NAD kinase, not NADH kinase. So this supposed transporter functions only as NADP transporter weakly during the mitochondrial NADPH hungry, not as NADPH transporter.

Finally, the detection of GFP-fused Yef1p and Utr1p might be successful by increasing the sensitivity of the fluorescence detection system, or by subcellular fractionation and then Western blot analysis using anti-GFP antibody.

Acknowledgement

We thank Prof. K. Murata and Dr. S. Kawai (Kyoto University, Kyoto, Japan) for helping us to perform this study.

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