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Cloning and characterization of vmaA, the gene encoding a 69-kDa catalytic subunit of the vacuolar H+-ATPase during alkaline pH mediated growth of Aspergillus oryzae
FEMS Microbiology Letters 209 (2002) 277^282
www.fems-microbiology.org
Cloning and characterization of vmaA, the gene encoding a 69-kDa catalytic subunit of the vacuolar Hþ -ATPase during alkaline pH mediated growth of Aspergillus oryzae Yutaka Kuroki, Praveen Rao Juvvadi, Manabu Arioka, Harushi Nakajima, Katsuhiko Kitamoto Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 November 2001; accepted 13 February 2002 First published online 11 March 2002
Abstract Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar Hþ -ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (vvma1) was viable at alkaline pH 8.0 and in the presence of CaCl2 (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vacuolar ATPase ; VMA1; vmaA; Alkaline pH; Aspergillus oryzae
1. Introduction Aspergillus oryzae, also known as the koji mold, has since long been exploited in the Japanese fermentative industry for fermentative processes involving the production of sake, soybean paste and soy sauce. Cultivation of pure tane-koji (conidia of A. oryzae) for the brewing of sake involves the addition of wood-ash in order to prevent any microbial contamination [1]. Inclusion of wood-ash causes a variation in pH of the medium from acidic or neutral towards alkalinity. Although A. oryzae adapts to a wide range of pH from 3 to 12, information on the expression of genes during alkaline pH adaptation of this ¢lamentous fungus remains obscure. Such an alteration in pH of the medium to alkalinity prompted us to investigate the expression of genes during alkaline pH mediated growth of A. oryzae. Acting as a primary storehouse for biological molecules,
* Corresponding author. Tel. : +81 (3) 58 41 51 61; Fax : +81 (3) 58 41 80 33. E-mail address : [email protected] (K. Kitamoto).
the fungal vacuole is a complex organelle involved in a plethora of functions ranging from assimilation and degradative processes to maintenance of intracellular homeostasis and pH regulation [2]. The vacuolar Hþ -ATPases (V-ATPases) are multi-enzyme complexes localized on the vacuolar membrane and can be structurally split into two major domains, namely the V1 peripheral domain harboring the ATP binding hydrolytic sector and the V0 transmembrane domain constituting the proton pore [3,4]. After the discovery of the ¢rst member of the V-ATPase family in 1981 in Saccharomyces cerevisiae, the complexity of this multisubunit enzyme began to be unraveled in other organisms including ¢lamentous fungi [2]. At least 13 genes encoding the various subunits of the multi-enzyme complex have been identi¢ed, of which eight genes encode proteins on the peripheral catalytic V1 subcomplex (subunits A^H of molecular mass 70^14 kDa ; responsible for ATP hydrolysis) and the remaining ¢ve genes encode proteins of the integral complex of molecular mass 100^17 kDa (subunits a, c, cP, cQ and d) responsible for proton translocation. V-ATPases generate an electrochemical gradient across the vacuolar membrane in order to provide the suitable chemical environment for maturation and
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 5 1 - 7
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functioning of the hydrolytic enzymes. Changes in the vacuolar lumen pH to a great extent e¡ect the targeting of soluble vacuolar proteins, implying vacuolar acidi¢cation as indispensable for e⁄cient sorting of proteins. In the light of their basic function in endomembrane energization and compartmentation, a putative role for V-ATPases during stress adaptation in ¢lamentous fungi has to be expected. V-ATPases, apart from being indispensable for growth in plants, have been proposed to facilitate the plant survival under various stress conditions of salinity, drought, cold, acid stress etc. [5], and in addition have been proposed to play a major role in pH adaptation and ion homeostasis in S. cerevisiae [6,7]. Therefore we chose the genes encoding V-ATPase from our data on the expressed sequence tags (ESTs) during alkaline pH mediated growth of A. oryzae for further analysis and cloned two V-ATPase genes homologous to vma1 (designated as vmaA) and vma3 (designated as vmaC; unpublished) from A. oryzae and herein report the functional characterization of the vmaA gene that is important for ion regulation and may provide an in-depth understanding of vital physiological processes of protein storage, transport and secretion during the alkaline pH stress response pathway in A. oryzae.
2. Materials and methods 2.1. Strains, plasmids, media and culture conditions The genomic DNA and cDNA libraries were derived from A. oryzae (RIB40). A. oryzae NS4 (sC3 ) was used for vmaA gene disruption. For cloning and subcloning experiments the phage vector V DASH II, Escherichia coli P2392 and pBluescript II SK+ vector were utilized. S. cerevisiae YOC1482 (ade2 lys2 his3 trp1 leu2 ura3 vvma1: :TRP1) (donated by Prof. Y. Ohya, University of Tokyo) served as a host for the expression of A. oryzae vmaA cDNA. The yeast expression vector pYES2 that contained a GAL1 promoter and a URA3 marker was used for the introduction of A. oryzae vmaA cDNA. S. cerevisiae was cultured in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) and YNBD medium (0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI, USA) and 2% glucose). For yeast complementation experiments YNBD medium plates supplemented with adenine, lysine, histidine and leucine at a concentration of 20 Wg ml31 (for selection of transformants) and YPGal (1% yeast extract, 2% polypeptone and 2% galactose; pH 6.5) medium alone or supplemented with 100 mM CaCl2 or at pH 8.0 and agar (2%) were used. The transformation of E. coli and S. cerevisiae was performed as described [8,9]. All standard molecular biological procedures were performed according to Sambrook et al. [10]. The sC gene encoding ATP-sulfurylase on pUSC was used as the selection marker for disruption of
the vmaA gene. Czapeks Dox (CD) medium (0.3% NaNO3 , 0.2% KCl, 0.1% KH2 PO4 , 0.05% MgSO4 W7H2 O, 0.002% FeSO4 W7H2 O and 2% glucose; pH 5.5), DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2 PO4 , 0.05% MgSO4 W7H2 O; pH 5.5), were used as minimal and complete medium, respectively, for culturing A. oryzae. M-Medium consisted of 0.2% NH4 Cl, 0.1% (NH4 )2 SO4 , 0.05% KCl, 0.05% NaCl, 0.1% KH2 PO4 , 0.05% MgSO4 W7H2 O, 0.002% FeSO4 W7H2 O, 2% glucose (pH 5.5). A. oryzae transformants were grown in M-medium supplemented with 100 mM CaCl2 or in M-medium adjusted to pH 10.0. 2.2. Strategy for the cloning and sequencing of A. oryzae vmaA In order to clone the vmaA encoding gene from A. oryzae, two oligonucleotide primers, vmc-f (5P-GGG AAT TCA TAA TGG CCC CCT CC-3P) and vmc-r (5P-GGG AAT TCT TAC TCA TCC GAC ACC-3P), were designed based on the vmaA cDNA nucleotide sequence of a clone obtained by EST analysis. A 2072-bp fragment ampli¢ed by PCR using A. oryzae genomic DNA as template and vmc-f and vmc-r primers was cloned using pBluescript II SK+ vector and sequenced to con¢rm its homology to vmaA related genes from other organisms. Among approximately 5U104 plaques of the DASH II genomic library screened using the 2072-bp PCR fragment as probe, eight positive clones were identi¢ed. Restriction enzyme digestion of the phage DNA followed by Southern analysis with the probe revealed a single 6-kb XbaI fragment that hybridized with the probe, which was subcloned using pBluescript II SK+ vector and its sequence was determined on a Shimadzu DSQ-2000L automated £uorescent sequencer. 2.3. Yeast complementation To facilitate the expression of A. oryzae vmaA in S. cerevisiae, the vmaA cDNA ampli¢ed by PCR using primers vmc-f and vmc-r and A. oryzae cDNA library as template was inserted into the EcoRI site on the 2 W yeast expression vector pYES2 under the control of GAL1 promoter to generate the plasmid pYESVA. The S. cerevisiae vma1 disrupted strain (YOC1482) was transformed with pYESVA. The transformants were selected as described in Section 2.1 and cultured in the presence of high concentrations of Ca2þ (100 mM CaCl2 ) and also under alkaline pH condition (pH 8.0) to verify the complementation of A. oryzae vmaA in the S. cerevisiae vma1 disrupted strain. 2.4. Northern analysis A. oryzae was grown in CD agar medium of pH 4.0, 7.0, and 10.0 at 30‡C for 72 h. The mycelia were homogenized
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using liquid nitrogen and total RNA was extracted [11]. RNA (10 Wg) was loaded on a 1% denaturing agarose gel and transferred onto Hybond Nþ membrane and ¢xed by UV illumination. The entire vmaA cDNA fragment labeled with [32 P]dCTP by the Random primer DNA Labeling kit (Takara Shuzo, Kyoto, Japan) was used as a hybridization probe.
2.6. Microscopic observation of A. oryzae vacuoles
2.5. vmaA gene disruption in A. oryzae
3. Results and discussion
A. oryzae vmaA gene was disrupted by constructing a pBluescript II SK+ based plasmid pBVA, and inserting the sC selectable marker gene at the unique KpnI site on the vmaA gene, generating pBVASC. The 8.5-kb XbaI fragment ampli¢ed by PCR was transformed into the A. oryzae NS4 strain (sC3 ). Transformants obtained on the selective M-medium, in which the resident vmaA gene had been replaced, were identi¢ed by Southern analysis using the 1.2-kb vmaA PCR ampli¢ed fragment using primers vmc-f and vmc-r2 (5P-GGG AAT TCC TGA GAA ATC ACC ACC-3P).
Although A. oryzae, an industrially important fungus, has been used as a source of useful enzymes especially in the Japanese food industry for a long time, its genetics is being investigated only recently. We have reported earlier the characterization of palBory from A. oryzae, a homolog of A. nidulans palB, and also a cysteine-like protease (CPL1) from S. cerevisiae involved in alkaline pH adaptation and sporulation [12,13]. In order to elucidate the pH stress mediated signal transduction pathway in A. oryzae we investigated the expression of genes in response to alkaline pH growth condition by constructing a cDNA
The A. oryzae strains were grown for 6 days on M-agar media at 30‡C, and the mycelia were observed with an Olympus AX80T microscope equipped with an automatic camera.
Fig. 1. Nucleotide and deduced amino acid sequences of the A. oryzae vmaA gene. The vmaA gene contains three introns, the positions of which were determined by the comparison with the cDNA sequence. The vmaA protein consists of 605 amino acids. The protein splicing consensus sequence is double underlined and the characteristic nine nucleotide residues are shown in bold. The ATP binding motifs are boxed. The putative PacC consensus binding site is indicated by an arrow. The sequence data reported in this paper have been submitted to the DDBJ nucleotide sequence database under accession No. AB073302.
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library under alkaline pH condition, which resulted in the identi¢cation of a gene encoding V-ATPase as a probable candidate involved in alkaline pH adaptation. Upon EST analysis a cDNA clone for vmaA was obtained and based on this sequence, cloning of the vmaA gene from A. oryzae was accomplished as described in Section 2.2. 3.1. Sequence analysis of vmaA gene from A. oryzae Nucleotide sequence analysis of vmaA genomic DNA and cDNA revealed an open reading frame (ORF) of 2072 bp and included three introns in contrast to four introns present in vma-1 from Neurospora crassa [14] and encoded a putative protein product of 605 amino acids, with a predicted molecular mass of 69 kDa (Fig. 1). Each intron had a 5P GT and 3P AG splice junction, characteristic of eukaryotic introns. The vmaA nucleotide sequence is deposited in the DDBJ nucleotide sequence database under accession No. AB073302. The deduced amino acid sequence revealed that a large portion of the ORF was homologous to Vma-1p from N. crassa (71%), Vma1p from S. cerevisiae (69%) and ATP6A2 from human (49%). In S. cerevisiae it has been reported that the characteristic nine nucleotide residues belonging to protein splicing sequences were present in the VMA1 gene (Fig. 1) resulting in the splicing of the translational product [15] and interestingly such consensus splicing sequences were also present in vmaA, although no mechanism of protein splicing is observed in this fungus. Further, an analysis of the promoter region of vmaA revealed the presence of a putative PacC binding consensus sequence positioned at 3149 bp upstream from the start codon. 3.2. Alkaline pH mediated growth of A. oryzae caused an enhanced expression of vmaA gene In order to analyze the expression of vmaA during growth of A. oryzae under varied pH conditions, the vmaA speci¢c mRNA levels were monitored by growing the organism in M-medium whose pH was adjusted to 4.0, 7.0 and 10.0. An induced and enhanced expression of vmaA was observed when A. oryzae was grown in alkaline
Fig. 2. Northern analysis of the vmaA gene under acidic, neutral and alkaline pH conditions in A. oryzae. Total RNA was separated on a 1% agarose, 0.6 M formaldehyde gel by electrophoresis (A), blotted onto Hybond Nþ membrane, and probed with 32 P labeled cDNA (B). The autoradiogram revealed the enhanced vmaA speci¢c mRNA levels under alkaline pH (10.0) mediated growth of A. oryzae.
Fig. 3. A. oryzae vmaA complemented the pH sensitivity of the S. cerevisiae vma1 disrupted strain. The 1815-bp EcoRI fragment encoding the A. oryzae vmaA cDNA was inserted into the EcoRI site of yeast expression plasmid pYES2, yielding pYESVA (A). The vma1 disrupted strain of S. cerevisiae (YOC 1482 ; vvma1) transformed with pYESVA was grown at 30‡C on bu¡ered YPGal medium plates containing 2% galactose as a carbon source at pH 6.5 and pH 8.0, respectively (B,C). The strains were also grown in the presence of 100 mM CaCl2 as described in Section 2 (D). The plates were incubated at 30‡C for 3 days. Plating of the transformants is as shown in the adjacent circle.
medium (pH 10.0). As shown in Fig. 2, although the expression of vmaA was observed under acidic condition (pH 4.0), it may be noted that the expression of the vmaA gene was very low at neutral pH 7.0, and subsequently enhanced when the organism was grown at an alkaline pH 10.0. Such an observation indicated an alkaline pH induced expression of vmaA supporting our data on EST analysis (data not shown), implicating a putative role for vmaA in alkaline pH mediated stress adaptation. 3.3. A. oryzae vmaA cDNA complemented the S. cerevisiae vma1 disrupted strain Since vmaA from A. oryzae revealed a highly conserved nature with respect to the A-subunit of V-ATPase from S. cerevisiae, the availability of a characteristic set of growth phenotypes of S. cerevisiae strains lacking the V-ATPase activity due to disruption of each of the VMA genes [16] has been exploited to verify if the A. oryzae vmaA could complement a null mutation of VMA1. As shown in Fig. 3, the vmaA cDNA when expressed under the control of the GAL1 promoter functionally complemented a S. cerevisiae V-ATPase disrupted strain. While the mutant phenotype exhibited sensitivity to increase in Ca2þ in the growth medium and inviability under neutral and alkaline pH conditions [17], the S. cerevisiae strain (YOC 1482 ; vvma1) transformed with pYESVA (Fig. 3A) inoculated onto YPGal induction medium (pH 6.5) in the presence or in the absence of 100 mM CaCl2 and onto YPGal medium (pH 8.0) was viable and therefore resistant to alkaline pH
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Fig. 4. Disruption of the vmaA gene from A. oryzae. Construction of the vmaA disrupted plasmid pBVASC. The 3.0-kb SmaI-EcoRV fragment containing the sC marker gene was introduced at the KpnI site on pBVA after blunting (A). Southern analysis of the vmaA disruptant. Genomic DNA from the wild-type, NS4, and vmaA disruptant, BVA, were digested with XbaI-SpeI and subjected to Southern analysis with the 1.2-kb vmaA probe (B,C).
condition. A transformant harboring only the vector pYES2 was selected as control. All transformants including the control transformant grew well by the end of 3 days on YPGal medium (pH 6.5) (Fig. 3B). However, at pH 8.0 (Fig. 3C) and in the presence of 100 mM CaCl2
(Fig. 3D) only the transformant containing vmaA cDNA could grow in comparison to the control transformant. Such a tolerant behavior of the transformant containing vmaA cDNA suggested a functional homology between A. oryzae vmaA and the yeast VMA1 genes.
Fig. 5. Growth of the A. oryzae vmaA disruptant on M-medium. The growth of the wild-type strain (NS4) and the vmaA disrupted strain (BVA) was examined under acidic, neutral and alkaline pH conditions (pH 5.0, 7.0, 8.5 and 10.0). Spores (1U103 ) were spotted on M-medium agar plate and the strains were grown at 30‡C for 6 days (A^D). The 6 day old mycelial cultures of the wild-type strain and the vmaA disrupted strain were examined by di¡erential interference microscopy as described in Section 2 (E^G). Scale bar, 10 Wm.
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3.4. Disruption of vmaA gene caused a pH dependent growth defect and abnormal vacuolar morphology After obtaining evidence on the functional homology between A. oryzae VmaAp and S. cerevisiae Vma1p, we transformed the A. oryzae NS4 strain with a plasmid, pBVASC, in which the vmaA gene was disrupted by the insertion of the sC gene as a selectable marker at the KpnI site of the vmaA gene (Fig. 4A) and generated a vmaA disrupted strain of A. oryzae. Southern analysis of vmaA from the wild-type strain and the vmaA disrupted strain, BVA, using the 1.2-kb fragment as a probe is shown in Fig. 4B,C. The BVA strain grew slowly on the M-medium agar plate (pH 5.0 and pH 7.0) in comparison with the wild-type strain (Fig. 5A,B). The BVA strain was examined for its ability to grow under alkaline pH conditions (Fig. 5C,D). Since yeast vma1 mutants were reported to be pH sensitive, we tested whether the BVA strain had similar sensitivity to the changes in acidic and alkaline growth conditions. While the di¡erential interference microscopic analysis showed normal vacuolar morphology in both the wild-type strain and the vmaA disruptant grown under acidic (Fig. 5E) and neutral pH conditions (Fig. 5F), the vmaA disrupted strain showed smaller or fragmented vacuoles and thin irregular hyphal walls, when grown at pH 8.5, implicating a probable role of vmaA for alkaline pH adaptation and also maintenance of the proper vacuolar structure (Fig. 5G). Compared to the wild-type strain, the BVA strain exhibited increased sensitivity to pH 8.5 (Fig. 5C) and could not grow at pH 10.0 (Fig. 5D). Moreover, the strain grew slowly under acidic pH conditions (Fig. 5A) but no changes in hyphal morphology were noted in either the wild-type strain or the vmaA disrupted strain, in contrast to the changes observed during alkaline pH mediated growth of the A. oryzae vmaA disrupted strain. Since any change in the acidity of the vacuolar compartment is known to cause abnormal function, studies on the storage and secretion of vacuolar proteins in the vmaA disrupted strain of A. oryzae would elucidate the consequence of vmaA deletion and also give more information on the function of V-ATPases during alkaline pH adaptation. In this regard, transformation of the vmaA disrupted strain of A. oryzae with a CPY-EGFP fusion protein (vacuolar carboxypeptidase and enhanced green £uorescent protein) is being carried out. Questions remain to be answered in relation to the speci¢c cellular signals that communicate extracellular pH changes to the intracellular VATPase complex and, more importantly, the characterization of individual enzyme activities of the V1 and V0 complex in A. oryzae may contribute to the knowledge on the regulation of V-ATPases in ¢lamentous fungi.
Acknowledgements This study was supported by a Grant-in-Aid for Scienti¢c Research (B) (No. 12460041) to K.K. from the Min-
istry of Education, Science, Sports, Technology and Culture, Japan. The authors thank Dr. Y. Ohya (University of Tokyo, Japan) for S. cerevisiae (YOC1482; vma1 disrupted strain).
References [1] Kitamoto, K., Gomi, K., Goto, K. and Hara, S. (1991) Genetic transfer applied to traditional sake brewing. Biotechnol. Genet. Eng. Rev. 9, 89^125. [2] Klionsky, D.J., Herman, P.K. and Emr, S.D. (1990) The fungal vacuole : composition, function, and biogenesis. Microbiol. Rev. 54, 266^ 292. [3] Bowman, B.J., Vazquez-Laslop, N. and Bowman, E.J. (1992) The vacuolar ATPase of Neurospora crassa. J. Bioenerg. Biomembr. 24, 361^370. [4] Forgac, M. (1999) Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases. J. Bioenerg. Biomembr. 31, 57^65. [5] Dietz, K.J., Tavakoli, N., Kluge, C., Mimura, T., Sharma, S.S., Harris, G.C., Chardonnens, A.N. and Golldack, D. (2001) Signi¢cance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J. Exp. Bot. 52, 1969^1980. [6] Nelson, H. and Nelson, N. (1990) Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality. Proc. Natl. Acad. Sci. USA 87, 3503^3507. [7] Yamashiro, C.T., Kane, P.M., Wolczyk, D.F., Preston, R.A. and Stevens, T.H. (1990) Role of vacuolar acidi¢cation in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol. Cell. Biol. 10, 3737^3749. [8] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 577^580. [9] Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163^168. [10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [11] Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156^159. [12] Futai, E., Sorimachi, H., Jeong, S.-Y., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) Aspergillus oryzae pal Bory encodes a calpain-like protease : homology to Emericella nidulans PalB and conservation of functional regions. J. Biosci. Bioeng. 88, 438^440. [13] Futai, E., Maeda, T., Sorimachi, H., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation. Mol. Gen. Genet. 260, 559^568. [14] Bowman, E.J., Tenney, K. and Bowman, B.J. (1988) Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases. J. Biol. Chem. 263, 13994^14001. [15] Kawasaki, M., Nogami, S., Satow, Y., Ohya, Y. and Anraku, Y. (1997) Identi¢cation of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence. J. Biol. Chem. 272, 15668^15674. [16] Nishikawa, S., Umemoto, N., Ohsumi, Y., Nakano, A. and Anraku, Y. (1990) Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae. J. Biol. Chem. 265, 7440^7448. [17] Anraku, Y. (1996) Handbook of Biological Physics, Vol. 2 (Konings, W.N., Kaback, H.R. and Lolkema, J.S., Eds.), pp. 93^109. Elsevier Science Publishing Co., New York.
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Cloning and characterization of vmaA, the gene encoding a 69-kDa catalytic subunit of the vacuolar Hþ -ATPase during alkaline pH mediated growth of Aspergillus oryzae Yutaka Kuroki, Praveen Rao Juvvadi, Manabu Arioka, Harushi Nakajima, Katsuhiko Kitamoto Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 5 November 2001; accepted 13 February 2002 First published online 11 March 2002
Abstract Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar Hþ -ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (vvma1) was viable at alkaline pH 8.0 and in the presence of CaCl2 (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Vacuolar ATPase ; VMA1; vmaA; Alkaline pH; Aspergillus oryzae
1. Introduction Aspergillus oryzae, also known as the koji mold, has since long been exploited in the Japanese fermentative industry for fermentative processes involving the production of sake, soybean paste and soy sauce. Cultivation of pure tane-koji (conidia of A. oryzae) for the brewing of sake involves the addition of wood-ash in order to prevent any microbial contamination [1]. Inclusion of wood-ash causes a variation in pH of the medium from acidic or neutral towards alkalinity. Although A. oryzae adapts to a wide range of pH from 3 to 12, information on the expression of genes during alkaline pH adaptation of this ¢lamentous fungus remains obscure. Such an alteration in pH of the medium to alkalinity prompted us to investigate the expression of genes during alkaline pH mediated growth of A. oryzae. Acting as a primary storehouse for biological molecules,
* Corresponding author. Tel. : +81 (3) 58 41 51 61; Fax : +81 (3) 58 41 80 33. E-mail address : [email protected] (K. Kitamoto).
the fungal vacuole is a complex organelle involved in a plethora of functions ranging from assimilation and degradative processes to maintenance of intracellular homeostasis and pH regulation [2]. The vacuolar Hþ -ATPases (V-ATPases) are multi-enzyme complexes localized on the vacuolar membrane and can be structurally split into two major domains, namely the V1 peripheral domain harboring the ATP binding hydrolytic sector and the V0 transmembrane domain constituting the proton pore [3,4]. After the discovery of the ¢rst member of the V-ATPase family in 1981 in Saccharomyces cerevisiae, the complexity of this multisubunit enzyme began to be unraveled in other organisms including ¢lamentous fungi [2]. At least 13 genes encoding the various subunits of the multi-enzyme complex have been identi¢ed, of which eight genes encode proteins on the peripheral catalytic V1 subcomplex (subunits A^H of molecular mass 70^14 kDa ; responsible for ATP hydrolysis) and the remaining ¢ve genes encode proteins of the integral complex of molecular mass 100^17 kDa (subunits a, c, cP, cQ and d) responsible for proton translocation. V-ATPases generate an electrochemical gradient across the vacuolar membrane in order to provide the suitable chemical environment for maturation and
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 5 1 - 7
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functioning of the hydrolytic enzymes. Changes in the vacuolar lumen pH to a great extent e¡ect the targeting of soluble vacuolar proteins, implying vacuolar acidi¢cation as indispensable for e⁄cient sorting of proteins. In the light of their basic function in endomembrane energization and compartmentation, a putative role for V-ATPases during stress adaptation in ¢lamentous fungi has to be expected. V-ATPases, apart from being indispensable for growth in plants, have been proposed to facilitate the plant survival under various stress conditions of salinity, drought, cold, acid stress etc. [5], and in addition have been proposed to play a major role in pH adaptation and ion homeostasis in S. cerevisiae [6,7]. Therefore we chose the genes encoding V-ATPase from our data on the expressed sequence tags (ESTs) during alkaline pH mediated growth of A. oryzae for further analysis and cloned two V-ATPase genes homologous to vma1 (designated as vmaA) and vma3 (designated as vmaC; unpublished) from A. oryzae and herein report the functional characterization of the vmaA gene that is important for ion regulation and may provide an in-depth understanding of vital physiological processes of protein storage, transport and secretion during the alkaline pH stress response pathway in A. oryzae.
2. Materials and methods 2.1. Strains, plasmids, media and culture conditions The genomic DNA and cDNA libraries were derived from A. oryzae (RIB40). A. oryzae NS4 (sC3 ) was used for vmaA gene disruption. For cloning and subcloning experiments the phage vector V DASH II, Escherichia coli P2392 and pBluescript II SK+ vector were utilized. S. cerevisiae YOC1482 (ade2 lys2 his3 trp1 leu2 ura3 vvma1: :TRP1) (donated by Prof. Y. Ohya, University of Tokyo) served as a host for the expression of A. oryzae vmaA cDNA. The yeast expression vector pYES2 that contained a GAL1 promoter and a URA3 marker was used for the introduction of A. oryzae vmaA cDNA. S. cerevisiae was cultured in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) and YNBD medium (0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI, USA) and 2% glucose). For yeast complementation experiments YNBD medium plates supplemented with adenine, lysine, histidine and leucine at a concentration of 20 Wg ml31 (for selection of transformants) and YPGal (1% yeast extract, 2% polypeptone and 2% galactose; pH 6.5) medium alone or supplemented with 100 mM CaCl2 or at pH 8.0 and agar (2%) were used. The transformation of E. coli and S. cerevisiae was performed as described [8,9]. All standard molecular biological procedures were performed according to Sambrook et al. [10]. The sC gene encoding ATP-sulfurylase on pUSC was used as the selection marker for disruption of
the vmaA gene. Czapeks Dox (CD) medium (0.3% NaNO3 , 0.2% KCl, 0.1% KH2 PO4 , 0.05% MgSO4 W7H2 O, 0.002% FeSO4 W7H2 O and 2% glucose; pH 5.5), DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2 PO4 , 0.05% MgSO4 W7H2 O; pH 5.5), were used as minimal and complete medium, respectively, for culturing A. oryzae. M-Medium consisted of 0.2% NH4 Cl, 0.1% (NH4 )2 SO4 , 0.05% KCl, 0.05% NaCl, 0.1% KH2 PO4 , 0.05% MgSO4 W7H2 O, 0.002% FeSO4 W7H2 O, 2% glucose (pH 5.5). A. oryzae transformants were grown in M-medium supplemented with 100 mM CaCl2 or in M-medium adjusted to pH 10.0. 2.2. Strategy for the cloning and sequencing of A. oryzae vmaA In order to clone the vmaA encoding gene from A. oryzae, two oligonucleotide primers, vmc-f (5P-GGG AAT TCA TAA TGG CCC CCT CC-3P) and vmc-r (5P-GGG AAT TCT TAC TCA TCC GAC ACC-3P), were designed based on the vmaA cDNA nucleotide sequence of a clone obtained by EST analysis. A 2072-bp fragment ampli¢ed by PCR using A. oryzae genomic DNA as template and vmc-f and vmc-r primers was cloned using pBluescript II SK+ vector and sequenced to con¢rm its homology to vmaA related genes from other organisms. Among approximately 5U104 plaques of the DASH II genomic library screened using the 2072-bp PCR fragment as probe, eight positive clones were identi¢ed. Restriction enzyme digestion of the phage DNA followed by Southern analysis with the probe revealed a single 6-kb XbaI fragment that hybridized with the probe, which was subcloned using pBluescript II SK+ vector and its sequence was determined on a Shimadzu DSQ-2000L automated £uorescent sequencer. 2.3. Yeast complementation To facilitate the expression of A. oryzae vmaA in S. cerevisiae, the vmaA cDNA ampli¢ed by PCR using primers vmc-f and vmc-r and A. oryzae cDNA library as template was inserted into the EcoRI site on the 2 W yeast expression vector pYES2 under the control of GAL1 promoter to generate the plasmid pYESVA. The S. cerevisiae vma1 disrupted strain (YOC1482) was transformed with pYESVA. The transformants were selected as described in Section 2.1 and cultured in the presence of high concentrations of Ca2þ (100 mM CaCl2 ) and also under alkaline pH condition (pH 8.0) to verify the complementation of A. oryzae vmaA in the S. cerevisiae vma1 disrupted strain. 2.4. Northern analysis A. oryzae was grown in CD agar medium of pH 4.0, 7.0, and 10.0 at 30‡C for 72 h. The mycelia were homogenized
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using liquid nitrogen and total RNA was extracted [11]. RNA (10 Wg) was loaded on a 1% denaturing agarose gel and transferred onto Hybond Nþ membrane and ¢xed by UV illumination. The entire vmaA cDNA fragment labeled with [32 P]dCTP by the Random primer DNA Labeling kit (Takara Shuzo, Kyoto, Japan) was used as a hybridization probe.
2.6. Microscopic observation of A. oryzae vacuoles
2.5. vmaA gene disruption in A. oryzae
3. Results and discussion
A. oryzae vmaA gene was disrupted by constructing a pBluescript II SK+ based plasmid pBVA, and inserting the sC selectable marker gene at the unique KpnI site on the vmaA gene, generating pBVASC. The 8.5-kb XbaI fragment ampli¢ed by PCR was transformed into the A. oryzae NS4 strain (sC3 ). Transformants obtained on the selective M-medium, in which the resident vmaA gene had been replaced, were identi¢ed by Southern analysis using the 1.2-kb vmaA PCR ampli¢ed fragment using primers vmc-f and vmc-r2 (5P-GGG AAT TCC TGA GAA ATC ACC ACC-3P).
Although A. oryzae, an industrially important fungus, has been used as a source of useful enzymes especially in the Japanese food industry for a long time, its genetics is being investigated only recently. We have reported earlier the characterization of palBory from A. oryzae, a homolog of A. nidulans palB, and also a cysteine-like protease (CPL1) from S. cerevisiae involved in alkaline pH adaptation and sporulation [12,13]. In order to elucidate the pH stress mediated signal transduction pathway in A. oryzae we investigated the expression of genes in response to alkaline pH growth condition by constructing a cDNA
The A. oryzae strains were grown for 6 days on M-agar media at 30‡C, and the mycelia were observed with an Olympus AX80T microscope equipped with an automatic camera.
Fig. 1. Nucleotide and deduced amino acid sequences of the A. oryzae vmaA gene. The vmaA gene contains three introns, the positions of which were determined by the comparison with the cDNA sequence. The vmaA protein consists of 605 amino acids. The protein splicing consensus sequence is double underlined and the characteristic nine nucleotide residues are shown in bold. The ATP binding motifs are boxed. The putative PacC consensus binding site is indicated by an arrow. The sequence data reported in this paper have been submitted to the DDBJ nucleotide sequence database under accession No. AB073302.
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library under alkaline pH condition, which resulted in the identi¢cation of a gene encoding V-ATPase as a probable candidate involved in alkaline pH adaptation. Upon EST analysis a cDNA clone for vmaA was obtained and based on this sequence, cloning of the vmaA gene from A. oryzae was accomplished as described in Section 2.2. 3.1. Sequence analysis of vmaA gene from A. oryzae Nucleotide sequence analysis of vmaA genomic DNA and cDNA revealed an open reading frame (ORF) of 2072 bp and included three introns in contrast to four introns present in vma-1 from Neurospora crassa [14] and encoded a putative protein product of 605 amino acids, with a predicted molecular mass of 69 kDa (Fig. 1). Each intron had a 5P GT and 3P AG splice junction, characteristic of eukaryotic introns. The vmaA nucleotide sequence is deposited in the DDBJ nucleotide sequence database under accession No. AB073302. The deduced amino acid sequence revealed that a large portion of the ORF was homologous to Vma-1p from N. crassa (71%), Vma1p from S. cerevisiae (69%) and ATP6A2 from human (49%). In S. cerevisiae it has been reported that the characteristic nine nucleotide residues belonging to protein splicing sequences were present in the VMA1 gene (Fig. 1) resulting in the splicing of the translational product [15] and interestingly such consensus splicing sequences were also present in vmaA, although no mechanism of protein splicing is observed in this fungus. Further, an analysis of the promoter region of vmaA revealed the presence of a putative PacC binding consensus sequence positioned at 3149 bp upstream from the start codon. 3.2. Alkaline pH mediated growth of A. oryzae caused an enhanced expression of vmaA gene In order to analyze the expression of vmaA during growth of A. oryzae under varied pH conditions, the vmaA speci¢c mRNA levels were monitored by growing the organism in M-medium whose pH was adjusted to 4.0, 7.0 and 10.0. An induced and enhanced expression of vmaA was observed when A. oryzae was grown in alkaline
Fig. 2. Northern analysis of the vmaA gene under acidic, neutral and alkaline pH conditions in A. oryzae. Total RNA was separated on a 1% agarose, 0.6 M formaldehyde gel by electrophoresis (A), blotted onto Hybond Nþ membrane, and probed with 32 P labeled cDNA (B). The autoradiogram revealed the enhanced vmaA speci¢c mRNA levels under alkaline pH (10.0) mediated growth of A. oryzae.
Fig. 3. A. oryzae vmaA complemented the pH sensitivity of the S. cerevisiae vma1 disrupted strain. The 1815-bp EcoRI fragment encoding the A. oryzae vmaA cDNA was inserted into the EcoRI site of yeast expression plasmid pYES2, yielding pYESVA (A). The vma1 disrupted strain of S. cerevisiae (YOC 1482 ; vvma1) transformed with pYESVA was grown at 30‡C on bu¡ered YPGal medium plates containing 2% galactose as a carbon source at pH 6.5 and pH 8.0, respectively (B,C). The strains were also grown in the presence of 100 mM CaCl2 as described in Section 2 (D). The plates were incubated at 30‡C for 3 days. Plating of the transformants is as shown in the adjacent circle.
medium (pH 10.0). As shown in Fig. 2, although the expression of vmaA was observed under acidic condition (pH 4.0), it may be noted that the expression of the vmaA gene was very low at neutral pH 7.0, and subsequently enhanced when the organism was grown at an alkaline pH 10.0. Such an observation indicated an alkaline pH induced expression of vmaA supporting our data on EST analysis (data not shown), implicating a putative role for vmaA in alkaline pH mediated stress adaptation. 3.3. A. oryzae vmaA cDNA complemented the S. cerevisiae vma1 disrupted strain Since vmaA from A. oryzae revealed a highly conserved nature with respect to the A-subunit of V-ATPase from S. cerevisiae, the availability of a characteristic set of growth phenotypes of S. cerevisiae strains lacking the V-ATPase activity due to disruption of each of the VMA genes [16] has been exploited to verify if the A. oryzae vmaA could complement a null mutation of VMA1. As shown in Fig. 3, the vmaA cDNA when expressed under the control of the GAL1 promoter functionally complemented a S. cerevisiae V-ATPase disrupted strain. While the mutant phenotype exhibited sensitivity to increase in Ca2þ in the growth medium and inviability under neutral and alkaline pH conditions [17], the S. cerevisiae strain (YOC 1482 ; vvma1) transformed with pYESVA (Fig. 3A) inoculated onto YPGal induction medium (pH 6.5) in the presence or in the absence of 100 mM CaCl2 and onto YPGal medium (pH 8.0) was viable and therefore resistant to alkaline pH
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Fig. 4. Disruption of the vmaA gene from A. oryzae. Construction of the vmaA disrupted plasmid pBVASC. The 3.0-kb SmaI-EcoRV fragment containing the sC marker gene was introduced at the KpnI site on pBVA after blunting (A). Southern analysis of the vmaA disruptant. Genomic DNA from the wild-type, NS4, and vmaA disruptant, BVA, were digested with XbaI-SpeI and subjected to Southern analysis with the 1.2-kb vmaA probe (B,C).
condition. A transformant harboring only the vector pYES2 was selected as control. All transformants including the control transformant grew well by the end of 3 days on YPGal medium (pH 6.5) (Fig. 3B). However, at pH 8.0 (Fig. 3C) and in the presence of 100 mM CaCl2
(Fig. 3D) only the transformant containing vmaA cDNA could grow in comparison to the control transformant. Such a tolerant behavior of the transformant containing vmaA cDNA suggested a functional homology between A. oryzae vmaA and the yeast VMA1 genes.
Fig. 5. Growth of the A. oryzae vmaA disruptant on M-medium. The growth of the wild-type strain (NS4) and the vmaA disrupted strain (BVA) was examined under acidic, neutral and alkaline pH conditions (pH 5.0, 7.0, 8.5 and 10.0). Spores (1U103 ) were spotted on M-medium agar plate and the strains were grown at 30‡C for 6 days (A^D). The 6 day old mycelial cultures of the wild-type strain and the vmaA disrupted strain were examined by di¡erential interference microscopy as described in Section 2 (E^G). Scale bar, 10 Wm.
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3.4. Disruption of vmaA gene caused a pH dependent growth defect and abnormal vacuolar morphology After obtaining evidence on the functional homology between A. oryzae VmaAp and S. cerevisiae Vma1p, we transformed the A. oryzae NS4 strain with a plasmid, pBVASC, in which the vmaA gene was disrupted by the insertion of the sC gene as a selectable marker at the KpnI site of the vmaA gene (Fig. 4A) and generated a vmaA disrupted strain of A. oryzae. Southern analysis of vmaA from the wild-type strain and the vmaA disrupted strain, BVA, using the 1.2-kb fragment as a probe is shown in Fig. 4B,C. The BVA strain grew slowly on the M-medium agar plate (pH 5.0 and pH 7.0) in comparison with the wild-type strain (Fig. 5A,B). The BVA strain was examined for its ability to grow under alkaline pH conditions (Fig. 5C,D). Since yeast vma1 mutants were reported to be pH sensitive, we tested whether the BVA strain had similar sensitivity to the changes in acidic and alkaline growth conditions. While the di¡erential interference microscopic analysis showed normal vacuolar morphology in both the wild-type strain and the vmaA disruptant grown under acidic (Fig. 5E) and neutral pH conditions (Fig. 5F), the vmaA disrupted strain showed smaller or fragmented vacuoles and thin irregular hyphal walls, when grown at pH 8.5, implicating a probable role of vmaA for alkaline pH adaptation and also maintenance of the proper vacuolar structure (Fig. 5G). Compared to the wild-type strain, the BVA strain exhibited increased sensitivity to pH 8.5 (Fig. 5C) and could not grow at pH 10.0 (Fig. 5D). Moreover, the strain grew slowly under acidic pH conditions (Fig. 5A) but no changes in hyphal morphology were noted in either the wild-type strain or the vmaA disrupted strain, in contrast to the changes observed during alkaline pH mediated growth of the A. oryzae vmaA disrupted strain. Since any change in the acidity of the vacuolar compartment is known to cause abnormal function, studies on the storage and secretion of vacuolar proteins in the vmaA disrupted strain of A. oryzae would elucidate the consequence of vmaA deletion and also give more information on the function of V-ATPases during alkaline pH adaptation. In this regard, transformation of the vmaA disrupted strain of A. oryzae with a CPY-EGFP fusion protein (vacuolar carboxypeptidase and enhanced green £uorescent protein) is being carried out. Questions remain to be answered in relation to the speci¢c cellular signals that communicate extracellular pH changes to the intracellular VATPase complex and, more importantly, the characterization of individual enzyme activities of the V1 and V0 complex in A. oryzae may contribute to the knowledge on the regulation of V-ATPases in ¢lamentous fungi.
Acknowledgements This study was supported by a Grant-in-Aid for Scienti¢c Research (B) (No. 12460041) to K.K. from the Min-
istry of Education, Science, Sports, Technology and Culture, Japan. The authors thank Dr. Y. Ohya (University of Tokyo, Japan) for S. cerevisiae (YOC1482; vma1 disrupted strain).
References [1] Kitamoto, K., Gomi, K., Goto, K. and Hara, S. (1991) Genetic transfer applied to traditional sake brewing. Biotechnol. Genet. Eng. Rev. 9, 89^125. [2] Klionsky, D.J., Herman, P.K. and Emr, S.D. (1990) The fungal vacuole : composition, function, and biogenesis. Microbiol. Rev. 54, 266^ 292. [3] Bowman, B.J., Vazquez-Laslop, N. and Bowman, E.J. (1992) The vacuolar ATPase of Neurospora crassa. J. Bioenerg. Biomembr. 24, 361^370. [4] Forgac, M. (1999) Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases. J. Bioenerg. Biomembr. 31, 57^65. [5] Dietz, K.J., Tavakoli, N., Kluge, C., Mimura, T., Sharma, S.S., Harris, G.C., Chardonnens, A.N. and Golldack, D. (2001) Signi¢cance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J. Exp. Bot. 52, 1969^1980. [6] Nelson, H. and Nelson, N. (1990) Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality. Proc. Natl. Acad. Sci. USA 87, 3503^3507. [7] Yamashiro, C.T., Kane, P.M., Wolczyk, D.F., Preston, R.A. and Stevens, T.H. (1990) Role of vacuolar acidi¢cation in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol. Cell. Biol. 10, 3737^3749. [8] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 577^580. [9] Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163^168. [10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [11] Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156^159. [12] Futai, E., Sorimachi, H., Jeong, S.-Y., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) Aspergillus oryzae pal Bory encodes a calpain-like protease : homology to Emericella nidulans PalB and conservation of functional regions. J. Biosci. Bioeng. 88, 438^440. [13] Futai, E., Maeda, T., Sorimachi, H., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation. Mol. Gen. Genet. 260, 559^568. [14] Bowman, E.J., Tenney, K. and Bowman, B.J. (1988) Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases. J. Biol. Chem. 263, 13994^14001. [15] Kawasaki, M., Nogami, S., Satow, Y., Ohya, Y. and Anraku, Y. (1997) Identi¢cation of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence. J. Biol. Chem. 272, 15668^15674. [16] Nishikawa, S., Umemoto, N., Ohsumi, Y., Nakano, A. and Anraku, Y. (1990) Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae. J. Biol. Chem. 265, 7440^7448. [17] Anraku, Y. (1996) Handbook of Biological Physics, Vol. 2 (Konings, W.N., Kaback, H.R. and Lolkema, J.S., Eds.), pp. 93^109. Elsevier Science Publishing Co., New York.
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