The Aspergillus nidulans xprF Gene Encodes a Hexokinase-like Protein Involved in the Regulation of Extracellular Proteases

Corresponding author: Margaret E. Katz, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia., mkatz{at}metz.une.edu.au (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG^- mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.

THE filamentous fungus Aspergillus nidulans can use exogenous protein as a source of carbon, nitrogen, or sulfur. Utilization of exogenous protein is dependent on the production of extracellular proteases. Two genes encoding extracellular proteases have been isolated from A. nidulans. The prtA gene encodes an alkaline/serine protease (KATZ et al. 1994 ). Disruption of the prtA gene showed that the prtA-encoded serine protease is the most abundant extracellular protease detected in a wide range of pH conditions (VANKUYK et al. 2000 ). A gene (prtB) encoding a putative acid/aspartic protease has also been isolated from A. nidulans (VANKUYK et al. 2000 ). It is likely that additional genes encoding extracellular proteases are present in A. nidulans as six types of extracellular protease genes have been isolated from other Aspergillus species (TATSUMI et al. 1989 , TATSUMI et al. 1991 ; BERKA et al. 1990 ; INOUE et al. 1991 ; SIRAKOVA et al. 1994 ; VAN DEN HOMBERGH et al. 1994 ).

In A. nidulans, the production of extracellular proteases is subject to at least four regulatory mechanisms—carbon, nitrogen, and sulfur metabolite repression and pH control. A fifth regulatory mechanism, induction by exogenous protein, is present in A. niger (JARAI and BUXTON 1994 ) and may also operate in A. nidulans under certain conditions. However, all of the A. nidulans extracellular proteases that have been detected are produced in the absence of exogenous protein if the fungus is exposed to carbon-, nitrogen-, or sulfur-limiting nutrient conditions (COHEN 1973 ).

A number of genes that may be involved, directly or indirectly, in the regulation of the extracellular proteases have been identified through genetic studies. Nitrogen metabolite repression of the A. nidulans extracellular proteases is mediated by the areA gene product, a positive-acting DNA-binding protein (COHEN 1972 ; KUDLA et al. 1990 ). A. nidulans strains carrying areA^- loss-of-function mutations are unable to use protein as a source of nitrogen (ARST and COVE 1973 ). Mutations in the creB and creC genes lead to higher levels of extracellular protease and to derepression of some enzymes involved in carbon catabolism (HYNES and KELLY 1977 ). The creA gene encodes a DNA-binding protein that plays a major role in carbon catabolite repression in A. nidulans, but it is not known whether the creB and creC genes have a direct involvement in gene regulation (KELLY 1994 ).

In a previous study (KATZ et al. 1996 ) specifically designed to isolate A. nidulans mutants with altered levels of extracellular protease, three additional genes (xprE, xprF, and xprG) involved in the regulation of extracellular proteases were identified. The xprE1 mutation results in a protease-deficient phenotype that, at least in the case of the prtA gene, is due to very low levels of protease mRNA. The isolation of xprE1 revertants led to the characterization of the xprF1, xprF2, and xprG1 mutations, which suppress the protease-deficient phenotype of xprE1 mutants.

The xprF1, xprF2, and xprG1 mutants display a number of phenotypic similarities: (1) the mutants all show increased levels of extracellular protease in response to carbon limitation and, to a much lesser extent, nitrogen limitation. (2) The ability of these mutants to utilize a number of nitrogen sources is affected. (3) The xprF1 and xprG1 mutations suppress the areA217 defect in the utilization of milk as both a carbon and nitrogen source but not as a nitrogen source alone. (4) The xprG1 mutation and, to a lesser extent, the xprF1 and xprF2 mutations show incomplete dominance. Here, we provide evidence for the interaction of these two genes by showing that mutations in xprG can suppress mutations in the xprF gene. We also report the isolation of the xprF gene and show that this gene encodes a hexokinase-like protein that appears to represent a new class in this family of proteins.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Aspergillus strains, media, growth conditions, and genetic techniques:
The A. nidulans strains used in this study are listed in Table 1. A. nidulans was cultured at 37° in the minimal medium described by COVE 1966 except that glucose was omitted from media that contained other carbon sources. For media that contained 1% skim milk as a nitrogen or carbon source, sodium deoxycholate (0.08%) was used to induce compact colony formation. The techniques used for genetic manipulation of A. nidulans have been described (CLUTTERBUCK 1974 ).

Transformation of A. nidulans:
The preparation and transformation of A. nidulans protoplasts have been described previously (TILBURN et al. 1983 ). All transformation experiments involved cotransformation with a second plasmid carrying a selectable marker—pAN222, which carries part of the A. nidulans prn gene cluster (HULL et al. 1989 ), or pL3, which carries the A. nidulans riboB gene (OAKLEY et al. 1987 ). Transformation recipient strains carried either the prn309 deletion mutation and/or the riboB2 mutation. The prn⁺ (pAN222) or ribo⁺ (pL3) transformants were selected on medium containing proline as a nitrogen source and on medium lacking the vitamin riboflavin, respectively. Transformants were subsequently replicated to medium containing milk as a sole carbon source and medium containing hypoxanthine as a nitrogen source to test for the presence of the xprF gene on the nonselected DNA. The proportion of prn⁺ or ribo⁺ transformants that were also xpr⁺ was 7% for cotransformations with L32F12 cosmid DNA (7 out of 96 MK142 transformants) and 7% for pMK370 plasmid DNA (7 xpr⁺ribo⁺ transformants out of 111 ribo⁺ transformants of MK142 and 10 xpr⁺ribo⁺ transformants out of 141 ribo⁺ transformants of MK82).

Isolation of the xprF gene:
The A. nidulans chromosome-specific cosmid library, which consisted of A. nidulans DNA inserted in the pWE15 and Lorist2 cosmid vectors (BRODY et al. 1991 ), was obtained from the Fungal Genetics Stock Center. Clones containing chromosome VII cosmids were pooled into groups of 24. Cosmid DNA from nine pools containing 203 Lorist2 clones were used together with the selectable plasmid pAN222 in cotransformation experiments that used the xprF1 recipient strain MK142. Two xprF⁺ transformants were obtained from one of the nine pools. The 11 cosmids within this pool were then tested individually and cosmid L32F12 was shown to generate xprF⁺ cotransformants from the MK142 recipient strain.

To localize the xprF gene within L32F12, deletion subclones were generated from L32F12 by partial HindIII digestion and recircularization using T4 DNA ligase. The deletion subclones were tested for the ability to generate xprF⁺ transformants from the xprF1 recipient strain MK142 in cotransformation experiments using the pL3 selectable plasmid. Additional subclones and cosmid clones adjacent to L32F12 on the A. nidulans physical map were also tested.

DNA sequencing and molecular techniques:
Standard techniques were used for DNA cloning, restriction enzyme digestion, Southern and Northern blot analysis, and DNA sequencing (SAMBROOK et al. 1989 ).

Extraction of Aspergillus DNA and RNA:
Genomic DNA was extracted from A. nidulans by the method of ANDRIANOPOULOS and HYNES 1988 . RNA was prepared using a procedure developed by REINERT et al. 1981 .

RT-PCR analysis:
The Access reverse transcriptase (RT)-PCR System (Promega, Madison, WI) was used for all RT-PCR analyses. The reactions used 50 ng of total RNA prepared from mycelium of A. nidulans strain MH97 that had been grown in minimal medium containing 1% glucose as a carbon source. The reactions were performed using three pairs of primers, which are shown in Fig 4: (1) MK106 and MK110, (2) MK107 and MK110, and (3) MK113 and MK108. For each primer pair, a control PCR experiment was performed using DNA template. The RT-PCR product obtained with MK107 and MK110 was cloned using the SureClone ligation kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and the DNA sequence was determined.

View larger version (90K): In this window In a new window Download PPT slide   Figure 1. Phenotype of xprF1 revertants. Extracellular protease production of MH97 (wild type), MK142 (xprF1), and two revertant strains, MK186 (xprF1 xprG2) and MK187 (xprF1 xprG3), on medium containing 1% skim milk as a sole source of carbon (C) or nitrogen (N) are shown in the top row. The growth of the strains on medium containing 10 mM hypoxanthine as a sole nitrogen source is shown in the lower plate. The full genotypes of the strains are given in Table 1. Aspergillus media and growth conditions are described in MATERIALS AND METHODS.

View larger version (4K): In this window In a new window Download PPT slide   Figure 2. Partial linkage map of chromosome VII showing the location of the xprF gene. Gene order and linkage distances in centimorgans (cM) were derived from the analysis of 192 segregants from a cross between MK218 and G715. The distance between benC and choA has been reported as 6 cM and the distance between amdA and cnxF as 13 cM according to CLUTTERBUCK 1993 .

View larger version (59K): In this window In a new window Download PPT slide   Figure 3. Southern blot analysis and extracellular protease production of MK142 transformants. Transformants were generated by cotransformation of an xprF1 riboB2 strain (MK142) with pMK370 (containing the xprF gene) and pL3 (containing the riboB gene). Lanes 1–7 contain SmaI-digested genomic DNA from seven ribo+ xpr+ transformants of MK142, lanes 8 and 9 contain genomic DNA from two ribo+ transformants that did not display an xpr+ phenotype, and lane 10 contains DNA from the recipient strain, MK142. The blot was probed with the 0.3-kb HindII fragment indicated under the restriction map of pMK370 (S, SmaI; P, PstI; H, HindIII). Transformants that have acquired a copy of pMK370 are expected to contain an additional 1.6-kb SmaI fragment. Extracellular protease production of the transformants, the xprF1 recipient strain MK142, and a wild-type strain (MH97), on medium containing 1% skim milk as a carbon source, is shown on the right.

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. DNA sequence of the A. nidulans sequences in pMK370 (EMBL/GenBank/DDBJ accession no. AJ251893). Restriction sites are marked in boldface type. The 5' GTRNGT-RCTRAC-YAG consensus sequences for fungal introns (RAMBOSEK and LEACH 1986 ) are underlined and the intron is in lowercase letters. A potential nuclear localization sequence is double underlined and four leucine heptad repeats are circled. The base pair substitutions found in the xprF1 and xprF2 mutants are indicated with arrows. The oligonucleotides used in RT-PCR experiments are overlined. The sequences of oligonucleotides MK110 and MK108 are complementary to the sequences shown in the diagram.

Characterization of the xprF1 and xprF2 alleles:
The entire coding region of the xprF1 and xprF2 alleles was amplified in PCR experiments that used two primer pairs shown in Fig 4: (1) MK106 and MK108 and (2) MK107 and MK108. The DNA sequences of the xprF1 and xprF2 coding regions were determined by direct sequence analysis of the PCR amplification product and by sequence analysis of PCR products, which had been cloned using the SureClone ligation kit (Amersham Pharmacia Biotech). The single base pair alterations that were detected in each allele were confirmed by sequencing both strands of the amplified DNA.

Enzyme assays:
The assays used to measure protease activity in A. nidulans growth medium were performed as described previously (KATZ et al. 1996 ). The method of RUIJTER et al. 1996 was used to measure the phosphorylation of glucose and fructose in A. nidulans cell extracts prepared from mycelia grown for 16 hr in medium containing 1% glucose as a carbon source and then transferred to fresh medium containing 1% glucose for 3 hr.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of xprF1 revertants:
Strains carrying the xprF1 mutation are easily distinguished by two methods. The xprF1 mutation has a negative effect on the utilization of a number of nitrogen sources, particularly hypoxanthine, and causes a high level of extracellular protease activity on medium containing milk as a carbon source (Fig 1). The latter phenotype results in the production of colonies surrounded by a clear halo, due to extracellular protease activity. The xprF2 and xprG1 mutations have similar phenotypic effects. Wild-type strains produce high levels of extracellular protease activity on medium containing milk as a nitrogen source but are not surrounded by a clear halo after 48 hr of growth on medium containing milk as a carbon source (Fig 1).

Two xprF1 revertants (MK186 and MK187), which showed strong growth on medium containing hypoxanthine, were isolated. These revertants both had pale conidia and also showed a loss of extracellular protease activity on medium containing milk as a carbon or nitrogen source (Fig 1). Genetic analysis showed that both revertants carried extragenic suppressors that were unlinked to the xprF1 mutation. The phenotype of strains carrying only the suppressor mutation was indistinguishable from the phenotype of strains carrying both the xprF1 and suppressor mutations. The suppressor mutations from MK186 and MK187 were mapped to chromosome VII by haploidization analysis. The xprF and xprG genes are both located on chromosome VII but are unlinked. To determine whether the suppressor mutations were alleles of the xprG gene, a strain (MK198) carrying the MK186 suppressor mutation was crossed to an xprG1 strain (MK86). Only segregants that did not have pale conidia were scored for growth on hypoxanthine medium and extracellular production on medium containing milk as a carbon source. All of the 819 segregants that were scored proved to have an xprG1 phenotype. The lack of wild-type segregants from this cross suggests that the suppressor mutation in MK186 is an allele of the xprG gene and has thus been designated xprG2.

Two lines of evidence indicate that the xprF1 suppressor in MK187 is also an allele of the xprG gene (xprG3): (1) in a cross between strain MK198, which carries xprG2, and MK187, no wild-type segregants were detected among the 434 segregants scored and (2) a diploid constructed from MK198 and MK187 displayed a phenotype that was identical to both haploid strains unlike diploids constructed between haploid strains carrying each suppressor mutation and wild-type haploids. The latter xprG2/xprG⁺ and xprG3/xprG⁺ diploids had levels of extracellular protease that were similar to wild-type strains on medium containing milk as a nitrogen source, indicating that the xprG2 and xprG3 mutations are recessive.

Interactions with cre genes:
To examine the interactions between the xprF1 mutation and mutations in the creA, creB, and creC genes, double mutants were constructed. Because the creA204, creB15, and creC27 mutations relieve carbon catabolite repression of genes involved in ethanol metabolism, strains carrying these mutations are more sensitive than cre⁺ strains to the presence of allyl alcohol in growth medium containing a repressing carbon source such as sucrose. The xprF1 mutant does not show increased sensitivity to allyl alcohol. Strains carrying the creB15 and creC27 mutation have elevated levels of extracellular protease activity and reduced growth on hypoxanthine medium but to a lesser degree than the xprF1 mutant. The creA204 mutation does not increase extracellular protease production, nor affect utilization of hypoxanthine. The creB15 xprF1 and creC27 xprF1 double mutants showed a more extreme phenotype than xprF1, creB15, or creC27 single mutants. The double mutants produced higher levels of extracellular protease on medium containing milk as a carbon source and grew more poorly on medium containing hypoxanthine as a nitrogen source. The creA204 xprF1 mutants showed no evidence of an additive effect for the two mutations. These results suggest that mutations in the creB and creC genes affect the same regulatory pathway as mutations in the xprF gene.

Mapping of the xprF and xprG genes:
Previous studies had shown that the xprF and xprG genes were both located on A. nidulans chromosome VII but were unlinked to each other (KATZ et al. 1996 ). Genetic mapping was used to determine the position of the xprF gene on chromosome VII (Fig 2). The xprG gene was mapped to within 20 map units of the phenB gene.

To determine if either of these two genes were alleles of genes known to have a role in carbon or nitrogen regulation, we looked to see if any such genes are found on chromosome VII. The sarA and sarB genes are thought to have a role in nitrogen regulation (POLKINGHORNE and HYNES 1975 ). Though both genes are located on chromosome VII, their positions on the chromosome are unknown. The sarA3 and sarB7 mutations suppress the strong growth of the areA102 gain-of-function mutant on nitrogen sources such as lysine and histidine. Neither the xprF or xprG gene appeared to be linked to sarA, but the xprG gene proved to be very tightly linked to sarB. Of 1149 segregants from a cross between strains MK192 (xprG1) and TBH-7 (sarB7), three proved to be recombinants, including one that displayed the high extracellular protease levels of an xprG1 mutant and reduced ability to utilize lysine as a nitrogen source characteristic of a sarB7 mutant. The recovery of a segregant that showed a xprG1sarB7 phenotype makes it unlikely that sarB7 and xprG1 are alleles of the same gene but this possibility cannot be ruled out.

Isolation of the xprF gene:
The xprF gene was isolated from the chromosome-specific A. nidulans cosmid library (BRODY et al. 1991 ) by cotransformation of an xprF1 strain (MK142) with DNA from pooled chromosome VII-specific cosmids and a plasmid containing a selectable A. nidulans marker. A single cosmid (L32F12) that complemented the xprF1 mutation was obtained. The xprF gene was localized within the cosmid in similar cotransformation experiments using partial deletion derivatives of L32F12, plasmid subclones of L32F12, and adjacent cosmid clones from the physical map of A. nidulans that had subsequently become available (PRADE et al. 1997 ). The smallest segment of L32F12 DNA, which was capable of generating xprF⁺ transformants from an xprF1 recipient strain, was the 2.3-kb fragment inserted in pMK370 (Fig 3). Transformants that contained a single copy of pMK370 displayed normal levels of extracellular protease on media containing milk as a carbon or nitrogen source. Transformants that contained more than one copy of pMK370 had reduced levels of extracellular protease relative to a wild-type strain and distinctly paler conidial coloring though not as marked as in the xprG2 and xprG3 mutants. The pMK370 plasmid was also able to generate xprF⁺ transformants from an xprF2 strain (MK82), thereby confirming that the xprF1 and xprF2 mutations were alleles of the same gene.

DNA sequence analysis of the xprF gene:
Sequence analysis revealed that pMK370 contained an open reading frame of 615 amino acids interrupted by a single intron, the boundaries of which were confirmed by RT-PCR (Fig 4). As the open reading frame ends 54 bp from the end of the pMK370 insert, it is possible that pMK370 does not contain the xprF polyadenylation signal. No significant similarity between the 2.3-kb DNA sequence of the fragment containing the xprF gene and the sequences in GenBank was detected. However, the deduced amino acid sequence showed similarity to a large number of hexokinases and glucokinases (aldohexose-specific hexokinases). Surprisingly, the putative XprF sequence showed no greater similarity to fungal hexokinases than to human or plant hexokinases (16–19% amino acid identity with fungal hexokinases, mammalian glucokinases, Arabidopsis thaliana hexokinases, Entamoeba histolytica hexokinases, and Schistosoma mansoni hexokinases).

The isolation of three Aspergillus hexokinase genes has been reported, one from A. nidulans (frA, RUIJTER et al. 1996 ) and two from A. niger (glkA and hxkA, PANNEMAN et al. 1996 , PANNEMAN et al. 1998 ). Only part of the sequence from the A. nidulans frA gene product is available (RUIJTER et al. 1996 ). Comparison of the partial FrA sequence to the deduced amino acid sequence of the xprF gene shows many differences and the frA gene does not map to A. nidulans chromosome VII. The XprF sequence shows 18% amino acid identity to the A. niger hexokinase, HxkA, and 17% to the A. niger glucokinase, GlkA. In contrast, HxkA showed over 50% amino acid identity to hexokinases from Saccharomyces cerevisiae, Kluyveromyces lactis, Schwanniomyces occidentalis, and Yarrowia lipolytica (PANNEMAN et al. 1998 ) and GlkA showed 39% amino acid identity with the S. cerevisiae glucokinase and Schizosaccharomyces pombe hexokinase 2 (PANNEMAN et al. 1996 ).

Phylogenetic analysis confirmed that the XprF sequence was not closely related to other fungal hexokinases (Fig 5). Although the XprF sequence does not cluster with other fungal hexokinases, it is more closely related to hexokinases as a group than to other sugar kinases. In these analyses, the fungal hexokinases of all types formed a separate group, as do mammalian and plant hexokinases, suggesting that the development of multiple hexokinases has occurred independently within each lineage (BORK et al. 1993 ).

View larger version (29K): In this window In a new window Download PPT slide   Figure 5. Phylogenetic tree of the A. nidulans XprF sequence and hexokinases from other species. The sources for the amino acid sequences are as follows: A. thaliana hexokinase I and II (JANG et al. 1997 ) and hexokinase-like protein (GenPept accession no. CAB38932); A. niger glucokinase (PANNEMAN et al. 1996 ) and hexokinase (PANNEMAN et al. 1998 ); bovine hexokinase I (GRIFFIN et al. 1991 ); E. histolytica hexokinase 1 and 2 (ORTNER et al. 1995 ); human hexokinase I (NISHI et al. 1988 ), II (DEEB et al. 1993 ), III (FURUTA et al. 1996 ), and glucokinase (STOFFEL et al. 1992 ); K. lactis hexokinase (PRIOR et al. 1993 ) and galactokinase (MEYER et al. 1991 ); mouse hexokinase I (ARORA et al. 1990 ), II (HEIKKINEN et al. 1999 ), sperm hexokinase (MORI et al. 1993 ), and glucokinase (ISHIMURA-OKA et al. 1995 ); P. falciparum hexokinase (OLAFSSON et al. 1992 ); rat hexokinase I (SCHWAB and WILSON 1989 ), II (THELEN and WILSON 1991 ), III (SCHWAB and WILSON 1991 ), and glucokinase (HUGHES et al. 1991 ); S. cerevisiae glucokinase (ALBIG and ENTIAN 1988 ), hexokinase PI (KOPETZKI et al. 1985 ), hexokinase pII (FROHLICH et al. 1985 ), and galactokinase (BAJWA et al. 1988 ); S. mansoni hexokinase (SHOEMAKER et al. 1995 ); S. pombe hexokinase 1 and 2 (PETIT et al. 1996 ); S. occidentalis hexokinase (ROSE 1995 ); Y. lipolytica hexokinase I (PETIT and GANCEDO 1999 ). The rooted tree was constructed using the EPROTDIST and EKITSCH programs in the PHYLIP computer package (FELSENSTEIN 1996 ) through the Australian National Genome Information Service.

Structure of XprF:
Most of the conserved amino acid residues, which are found in the ATP-binding sites and sugar-binding sites of all other eukaryotic hexokinases, are present in the predicted xprF gene product (Fig 6). The putative hexose-binding site of XprF (GITFSFP) differs from the hexose-binding sites in most organisms, which contain the sequence GFTFSF/YP. The only other exceptions are the two hexokinases from E. histolytica (GLTFSFA). In addition, all other reported hexokinase sequences contain the sequence WTKGF seven amino acids from the C-terminal side of the sugar-binding site, whereas XprF contains the sequence MGKGF (Fig 6).

View larger version (101K): In this window In a new window Download PPT slide   Figure 6. Alignment of the putative XprF amino acid sequence with the sequences of the A. niger glucokinase (A.nig.glk; PANNEMAN et al. 1996 ) and hexokinase (A.nig.hex; PANNEMAN et al. 1998 ); the S. cerevisiae glucokinase (S.cer.glk; ALBIG and ENTIAN 1988 ), hexokinase PI (S.cer.hex1; KOPETZKI et al. 1985 ), and hexokinase PII (S.cer.hex2; FROHLICH et al. 1985 ); human glucokinase (H.sap.glk; STOFFEL et al. 1992 ); and A. thaliana hexokinase 1 (A.tha.hex; JANG et al. 1997 ). Amino acids that are conserved in all eight proteins are shaded in black, those conserved in at least six of the proteins are shaded in dark gray, and those conserved in at least half of the proteins are shaded in light gray. The ATP-binding sites and sugar-binding sites, identified by BORK et al. 1993 , are boxed. The alignment was generated with the ECLUSTALW (THOMPSON et al. 1994 ) and the GeneDoc (NICHOLAS et al. 1997 ) computer programs.

The XprF sequence is longer than other fungal hexokinases that have been characterized, the longest of which is the Y. lipolytica hexokinase at 534 amino acids (PETIT and GANCEDO 1999 ). The N terminus of the XprF protein contains 37 amino acid residues not found in other hexokinases. Fifteen of these 37 amino acids are acidic. Similarity to other hexokinases commences at a second methionine residue. RT-PCR experiments showed that xprF mRNA includes the sequences encoding this acidic region. Four leucine residues, at 7-amino-acid intervals, are found after this region (Fig 4). A potential nuclear localization sequence (KALDERON et al. 1984 ) is found at amino acids 256–262 (Fig 4). The XprF protein contains a stretch of some 60 amino acids in the C-terminal part of the protein that are not found in other hexokinases and interrupts part of the ATP-binding site (Fig 6).

Molecular characterization of the xprF1 and xprF2 mutations:
The nature of the xprF1 and xprF2 mutations was determined by amplification and sequence analysis of the xprF coding region from each mutant. A single base pair alteration was detected at nucleotide 1168 in the xprF1 allele and at nucleotide 1225 in the xprF2 allele. In both cases the base pair substitutions cause nonsense mutations (Fig 4).

Hexokinase activity in xprF^- mutants:
Since sequence analysis of the xprF gene suggested that it encodes a putative hexokinase, the xprF1 and xprF2 mutants were assayed for the ability to phosphorylate glucose and fructose (Table 2). The assays were performed in the presence and absence of trehalose 6-phosphate, which is an inhibitor of hexokinases but not glucokinases. Thus, the level of glucose phosphorylation activity observed, when trehalose 6-phosphate is present, is an indication of glucokinase activity. No differences between the mutant and wild-type activity levels were detected.

Sequence analysis of the A. nidulans frA gene indicates that it encodes a hexokinase and the frA1 mutant has been shown to have very low levels of fructose phosphorylating activity (RUIJTER et al. 1996 ). The residual fructose phosphorylating activity observed in the frA1 mutant was not abolished in an frA1xprF1 double mutant.

The effect of the xprF1 mutation on carbon catabolite repression of extracellular proteases:
Hexokinases are thought to play a role in triggering glucose repression in fungi (RONNE 1995 ). Thus, the most likely explanation for the phenotype of the xprF1 and xprF2 mutants is that the loss of the xprF-encoded hexokinase alleviates glucose repression of the extracellular proteases. However, production of extracellular proteases appeared to be completely repressed in mycelia transferred to medium containing glucose, lactose, galactose, or glycerol (Table 3) or in mycelia grown from conidia inoculated directly into media containing these carbon sources (data not shown). The effect of the xprF1 mutation on prtA mRNA levels was examined using Northern blot analysis. In medium containing glucose or glycerol, prtA expression was detected in the xprF1 mutant but not in a wild-type strain (Fig 7). However, the level of prtA expression was very much lower than is observed in carbon-limiting conditions.

View larger version (39K): In this window In a new window Download PPT slide   Figure 7. Northern blot analysis of alkaline protease mRNA levels. Total RNA was prepared from a wild-type (MH97) and xprF1 (MK142 and MK117) strains transferred to medium containing glucose, glycerol, or no carbon source for 16 hr. Duplicate blots were probed with 32P-labeled pBC174 plasmid containing the entire coding regions of the prtA gene (KATZ et al. 1994 ) and, as a control, a plasmid containing nucleotides 404–1711 of the constitutively expressed -actin gene (FIDEL et al. 1988 ). Note that much less RNA was used in the first lane.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The isolation and characterization of the xprF gene has shown that it encodes a new type of fungal hexokinase or hexokinase-like protein. Hexokinases catalyze the first step in the metabolism of glucose and other hexose sugars. In addition, hexokinases have been shown to have a role in the uptake of glucose (BISSON and FRAENKEL 1983 ) and as the glucose sensor that triggers glucose repression in fungi, plants, and animals.

The hexokinases of the yeast S. cerevisiae have been well characterized. Three genes (HXK1, HXK2, and GLK1) that encode enzymes for glucose phosphorylation are found in S. cerevisiae. A functional copy of only one of the three genes is required for glucose utilization (MAITRA and LOBO 1983 ). The HXK2 gene product is the predominant hexokinase and plays a major role in triggering glucose repression (ENTIAN 1980 ). However, there is some evidence that all three genes may be involved in mediating an early glucose repression response (SANZ et al. 1996 ). Recent work suggests that the S. cerevisiae hexokinase pII may have a direct role in regulating gene expression. It has been shown that nuclear localization of the S. cerevisiae hexokinase pII protein is required to trigger glucose repression of the SUC2 gene and hexokinase pII is a component of a DNA-protein complex that forms on SUC2 sequences (HERRERO et al. 1998 ).

Two hexokinase genes have been isolated from the plant A. thaliana by complementation in a S. cerevisiae hxk1hxk2 double mutant (JANG et al. 1997 ). The results of transformation experiments with heterologous hexokinase genes suggest that in plants and fungi (1) heterologous hexokinases are unable to activate the signal transduction pathway and (2) hexokinase catalytic activity alone is not sufficient for regulatory function (PRIOR et al. 1993 ; JANG et al. 1997 ). In mammals, the situation is different; glucose metabolism is required at least for some aspects of the glucose response (GERMAN 1993 ).

No regulatory role for hexokinases has been established in Aspergillus. The A. nidulans frA gene encodes the major hexokinase found in this fungus. In contrast to the situation in S. cerevisiae, loss of hexokinase activity in the frA1 mutant does not appear to affect glucose repression (RUIJTER et al. 1996 ).

The similarity of the xprF gene product to hexokinases suggests that it may be involved in carbon catabolite repression of the extracellular proteases. The xprF1 and xprF2 mutations have a clear effect on extracellular protease production as measured on medium containing milk as a sole source of carbon. Yet, in enzyme assays of the xprF1 mutant, the production of extracellular proteases appears to be fully repressed by glucose, lactose, galactose, and glycerol and only marginal derepression of the alkaline protease gene prtA was detected through Northern blot analysis. This discrepancy may be due to the presence of exogenous protein in milk, which could act as an inducer under these conditions, or it may reflect a greater sensitivity of milk plates in detecting low levels of extracellular protease.

Extracellular proteases are produced only if no source of carbon other than protein is present. Thus, the production of extracellular proteases may be a response to carbon starvation, which may involve a regulatory mechanism that is distinct from carbon catabolite repression. The xprF1 and xprF2 mutants exhibit higher levels of extracellular protease in response to carbon-limiting nutrient conditions suggesting that the xprF gene may have a role in the response to carbon starvation.

The demonstration that mutations in the xprG gene can suppress mutations in xprF provides further evidence for the interaction of these two genes. The data presented in this article are consistent with a model in which the xprG gene is a positive regulator of extracellular protease production and XprF prevents activation of extracellular protease gene expression by XprG, possibly through direct protein-protein interaction of the xprF and xprG gene products. The xprF1 and xprF2 mutations are both predicted to lead to the production of a truncated protein lacking the putative nuclear localization sequence. Thus, in the xprF1 and xprF2 mutants, activation of protease gene expression by XprG could not be blocked by XprF as no XprF protein would be present in the nucleus. Similarly, the reduced levels of extracellular protease in transformants containing more than one copy of the wild-type xprF gene could be due to increased inhibition of XprG activity.

If this model is correct, the xprG2 and xprG3 mutations would represent loss-of-function mutations in the positively acting xprG gene and the xprG1 mutation would represent a gain-of-function mutation, which might prevent the regulation of XprG by XprF. Three lines of evidence support this hypothesis. (1) The xprG1 mutation, which leads to increased extracellular protease activity, is partially dominant as would be expected of a gain-of-function mutation. (2) The xprG2 and xprG3 protease-deficient phenotype is recessive, which is consistent with loss-of-function mutations. (3) An xprG1/xprG2 diploid resembles an xprG1 haploid strain in phenotype. In addition, we have preliminary evidence that xprG1 revertants, which are identical in phenotype to the xprG2 and xprG3 protease-deficient mutants, carry a second mutation in the xprG gene.

The xprF1, xprF2, and xprG1 mutations were first identified as suppressors of the recessive xprE1 mutation, which leads to a protease-deficient phenotype. It has been proposed that the xprE gene could encode a protease-specific, positive-acting, regulator of extracellular protease production (KATZ et al. 1996 ). The xprE gene product could be an antagonist of XprF, preventing XprF-mediated inhibition of protease production. Alternatively, the xprE gene may be involved in the activation of a separate regulatory circuit, the loss of which is circumvented by the activation of XprG. An understanding of the roles of the xprG and xprE genes in the regulation of extracellular protease production will depend on the isolation and molecular characterization of these two genes.

Some of the phenotypic effects of xprF1 and xprF2 mutations are not completely recessive, which is unusual for loss-of-function mutations, but could be explained if the concentration of XprF in the nucleus is critical or if the truncated XprF1 and XprF2 proteins exert a dominant-negative effect through protein-protein interactions. The close proximity of the xprF1 and xprF2 nonsense mutations may not be a coincidence.

Mutations in both the xprF and xprG genes can affect nitrogen source utilization as well as extracellular protease production. The phenotype of the xprF1 and xprF2 mutants suggests a link between hexokinases and nitrogen regulation. In plants, sugars are known to affect many processes including nitrogen metabolism (LAM et al. 1994 ). In A. nidulans, a phenomenon that has been called reverse carbon catabolite repression leads to the repression of some enzymes involved in nitrogen metabolism under conditions of carbon starvation (DAVIS and HYNES 1991 ). Thus mutations in the xprF and xprG genes may affect enzymes that participate in nitrogen metabolism, if these genes are involved in triggering the response to carbon starvation.

The predicted XprF1 and XprF2 truncated proteins lack highly conserved hexokinase functional domains and would be nonfunctional with respect to sugar phosphorylating activity, yet no reduction in hexokinase or glucokinase activity was detected in the xprF1 and xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase that is expressed at low levels or (2) the xprF gene may encode a protein with no hexose phosphorylating activity.

The xprF gene product contains some unusual structural features. Plant genes encoding atypical hexokinase-like proteins have been identified (GenBank accession nos. CAB38932 and CAA63966). These putative proteins lack some of the consensus sequences in the sugar- and ATP-binding sites of hexokinases, so these genes may have a primarily regulatory function. It seems likely that additional atypical hexokinase-like proteins will be brought to light through genome sequencing projects.

	FOOTNOTES

¹ Present address: Division of Entomology, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia.

	ACKNOWLEDGMENTS

We gratefully acknowledge Ron Wicks for technical assistance, M. J. Hynes, M. A. Davis, and A. J. Clutterbuck for providing A. nidulans strains, and the Australian Research Council for supporting this project.

Manuscript received March 21, 2000; Accepted for publication July 21, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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