Original Paper
|
|
|
file on Synergy |
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
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.
References
1 Shi F, Kawai S, Mori S, Kono E, Murata K.
Identification of ATP-NADH kinase isozymes and their contribution to supply of
NADP(H) in Saccharomyces cerevisiae. FEBS J 2005, 272: 3337–3349
2 Outten CE, Culotta VC. A novel NADH kinase is
the mitochondrial source of NADPH in Saccharomyces cerevisiae. EMBO J
2003, 22: 2015–2024
3 Strand MK, Stuart GR, Longley MJ, Graziewicz
MA, Dominick OC, Copeland WC. POS5 gene of Saccharomyces cerevisiae
encodes a mitochondrial NADH kinase required for stability of mitochondrial
DNA. Eukaryot Cell 2003, 2: 809–820
4 Bannai H, Tamada Y, Maruyama O, Nakai K,
Miyano S. Extensive feature detection of N-terminal protein sorting signals.
Bioinformatics 2002, 18: 298–305
5 Wach A, Brachat A, Alberti-Segui C, Rebischung
C, Philippsen P. Heterologous HIS3 marker and GFP reporter modules for
PCR-targeting in Saccharomyces cerevisiae. Yeast 1997, 13: 1065–1075
6 Longtine MS, McKenzie A 3rd, Demarini DJ,
Shah NG, Wach A, Brachat A, Philippsen P et al. Additional modules for
versatile and economical PCR-based gene deletion and modification in Saccharomyces
cerevisiae. Yeast 1998, 14: 953–961
7 Gietz RD, Woods RA. Transformation of yeast
by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
Methods Enzymol 2002, 350: 87–96
8 Gietz RD, Sugino A. New yeast-Escherichia
coli shuttle vectors constructed with in vitro mutagenized yeast
genes lacking six-base pair restriction sites. Gene 1988, 74: 527–534
9 Tsien RY. The green fluorescent protein. Annu
Rev Biochem 1998, 67: 509–544
10 Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson
RW, Weissman JS, OShea EK. Global analysis of protein localization in budding
yeast. Nature 2003, 425: 686–691
11 Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle
A, Dephoure N, OShea EK et al. Global analysis of protein expression in
yeast. Nature 2003, 425: 737–741