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. 2005 Sep;171(1):393–401. doi: 10.1534/genetics.105.044644

Mutational Analysis of the pH Signal Transduction Component PalC of Aspergillus nidulans Supports Distant Similarity to BRO1 Domain Family Members

Joan Tilburn *,1, Juan C Sánchez-Ferrero †, Elena Reoyo †, Herbert N Arst Jr *, Miguel A Peñalva †
PMCID: PMC1456523  PMID: 15944343

Abstract

The alkaline ambient pH signal transduction pathway component PalC has no assigned molecular role. Therefore we attempted a gene-specific mutational analysis and obtained 55 new palC loss-of-function alleles including 24 single residue substitutions. Refined similarity searches reveal conserved PalC regions including one with convincing similarity to the BRO1 domain, denoted PCBROH, where clustering of mutational changes, including PCBROH key residue substitutions, supports its structural and/or functional importance. Since the BRO1 domain occurs in the multivesicular body (MVB) pathway protein Bro1/Vps31 and also the pH signal transduction protein PalA (Rim20), both of which interact with MVB component (ESCRT-III protein) Vps32/Snf7, this might reflect a further link between the pH response and endocytosis.

REGULATION by ambient pH has been extensively studied in Aspergillus nidulans where it is mediated by the PacC/Pal regulatory circuit and major contributions have also been made by studies on the equivalent Rim systems in the yeasts Saccharomyces cerevisiae, Candida albicans, and Yarrowia lipolytica.

Response to ambient pH in A. nidulans (reviewed by Peñalva and Arst 2002, 2004; Arst and Peñalva 2003) is mediated by PacC (Caddick et al. 1986; Tilburn et al. 1995), which is activated by a two-step proteolysis of the full-length form, PacC72 (Orejas et al. 1995; Mingot et al. 1999; Díez et al. 2002), in response to the alkaline ambient pH signal transduced by the six-membered Pal signaling pathway (Arst et al. 1994). The functional PacC 250-residue form, PacC27, is an activator of alkaline-expressed genes (Espeso and Peñalva 1996) and repressor of acid-expressed genes (Espeso and Arst 2000).

Signal transduction components PalH and PalI (S. cerevisiae homologs Rim21p and Rim9p, respectively) are predicted seven- and four-pass membrane proteins (Li and Mitchell 1997; Denison et al. 1998; Negrete-Urtasun et al. 1999) and strong candidates as ambient pH sensors. PalB (S. cerevisiae Rim13p), a calpain-like cysteine protease (Denison et al. 1995; Lamb et al. 2001; Sorimachi and Suzuki 2001), is probably responsible for the first, pH-sensitive, signaling proteolysis. PalA (S. cerevisiae Rim20p) (Negrete-Urtasun et al. 1997; Xu and Mitchell 2001) contains the ∼160-residue BRO1 domain (PFAM domain PF03097; http://www.sanger.ac.uk/Software/Pfam/index.shtml), first identified in yeast Bro1p (Nickas and Yaffe 1996). PalA apparently enables the signaling proteolysis by interacting both with PacC, through two YPXL/I motifs flanking the signaling proteolysis site, and with Vsp32/Snf7, the endosomal sorting complex required for transport-III (ESCRT-III) protein, as demonstrated by Vincent et al. (2003). This agrees with the model described for S. cerevisiae (Xu and Mitchell 2001; Xu et al. 2004) where Rim20p (PalA) interacts with both Rim101p (PacC) (Xu and Mitchell 2001) and Vsp32p/Snf7p, which also interacts with Rim13p (PalB) (Ito et al. 2001), to form a scaffold-promoting interaction between the Rim101p cleavage site and the Rim13p protease. A functional link between pH signal transduction and multivesicular body (MVB) pathway sorting complexes has been firmly established in S. cerevisiae (Xu et al. 2004) and shown to be conserved in C. albicans (Kullas et al. 2004; Xu et al. 2004).

Possible molecular roles remain elusive for PalF (S. cerevisiae Rim8p) and PalC, which has no S. cerevisiae homolog (Li and Mitchell 1997; Maccheroni et al. 1997; Negrete-Urtasun et al. 1999). As the PalC primary amino acid sequence revealed no evident sequence signature and PalC appeared absent from the hemiascomycete lineage, we carried out mutational analysis of this protein along with sequence profile similarity searching, exploiting the recent publication of a number of fungal genomes.

Mutational analysis:

The GABA (γ-aminobutyrate) technique is a powerful tool for the selection of pacC (Mingot et al. 1999; Fernández-Martínez et al. 2003) and pal (Arst et al. 1994; Denison et al. 1998; Negrete-Urtasun et al. 1999) loss-of-function mutations. It relies on the ability of these acidity-mimicking mutations to suppress areAr (= areA−, nitrogen metabolite repressed) mutations for utilization of γ-aminobutyrate (GABA) as the nitrogen source, through derepression of acid-expressed gabA specifying the GABA permease (Caddick et al. 1986; Hutchings et al. 1999; Espeso and Arst 2000). In haploid strains, the GABA technique yields mutations in any of the seven pH regulatory genes. To target mutations to palC, we employed an areAr palC+/areAr palC− diploid. This diploid cannot use GABA as the nitrogen source but suppression can be achieved by acidity mimicry, resulting from mutation of the palC+ allele or through mitotic recombination yielding palC− homozygosity. To avoid the latter, we constructed diploid R using inoB2 (inositol auxotrophy) distal to palC40 (Negrete-Urtasun et al. 1999) and in repulsion to areAr18 (Arst et al. 1989), a reciprocal translocation of chromosomes III and IV, including palC and inoB (Figure 1). Consequently, palC40 homozygosity without inositol auxotrophy can occur only through alignment of the translocation-containing chromosome III and the untranslocated chromosome IV with recombination both between the areAr18 breakpoint and palC40 (4 cM) and between palC40 and inoB2 (22 cM) (Figure 1).

Figure 1.

Figure 1.

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Diploid R (used for palC mutant selection). (A) Chromosomes III and IV of diploid R areAr18/areAr3 palC40 inoB2. The diploid was constructed using standard classical genetic techniques (Clutterbuck 1993) between parents of the relevant partial genotypes areAr18 (nitrogen metabolite repressed) (Arst et al. 1989) and inoB2 (inositol requiring), areAr3 (nitrogen metabolite repressed), and palC40 (acidity mimicking) (Negrete-Urtasun 1997; Negrete-Urtasun et al. 1999). (B) Chromosomes III and IV after replication. (Ci) If alignment and recombination occur between duplicated homologous (with respect to the centromeres) non-sister chromatids of chromosome IV, homozygosity for palC40 would always result in homozygosity for inoB2 and thus in inositol auxotrophy, due to the areAr18 translocation that precludes recombination between palC40 and inoB2. Recovery of palC40 inoB2 homozygotes is prevented by excluding inositol from the selection medium. (Cii) If alignment occurs between the homologous regions of chromosomes III and IV (with respect to the centromeres) and if two recombination events occur, one between the areAr18 breakpoint and palC40 and a second between palC40 and inoB2, inoB+ palC40 homozygotes can result.

The first experiment Table 1 yielded 48 acidity-mimicking—as determined by impaired growth on pH 8 medium (Cove 1976)—palC mutants. These included 24 new truncations, 8 single and one double missense, two with three-base deletions, and one rearrangement mutation (data not shown) plus 12 palC40 mitotic recombinants, despite precautions. In a second attempt and to avoid null phenotype palC truncations, we screened for leaky or temperature-sensitive growth on pH 8.0 medium and obtained another 20 palC missense mutants, thus totaling 55 new palC alleles (Table 1). Alignment (Figure 2) illustrates that most of the missense mutations affect amino acids conserved in the majority of the ascomycete PalCs shown. Of the single residue change mutations, only palC80 (Gly321Asp) and palC162 (ΔArg442) are complete loss-of-function mutations. All truncating mutations result in complete loss of function except the leaky palC131 (1–454 + 2), which removes 53 C-terminal residues including a completely conserved C-terminal di-aromatic motif. This motif (di-tyrosine in A. nidulans PalC) resembles the di-phenylalanine motif, a C-terminal transport motif facilitating ER export (Nufer et al. 2002). As palC131 is partially functional, this motif and other residues C-terminal to residue 454 cannot be completely essential for PalC structure and function. The complete loss-of-function truncating mutations palC159 (1–427 + 29) and palC153 and palC179, truncating the protein cleanly after residue 426, indicate that at least some of residues 427–454 are essential, which agrees with the clustering of mutations in this window. Mutations substituting conserved residues in regions “BRO1 similar,” “LALA,” and “ERRE” (Figure 4B) further support the structural and/or functional importance of these regions. We caution, however, that the phenotypes of any of the palC mutations characterized here might be due to reduced PalC protein levels resulting from protein misfolding with consequent instability or even from messenger instability rather than from PalC dysfunction.

TABLE 1.

palC mutations isolated in this work

Allele Phenotype pH 8.0 Nucleotide change(s) Change in protein Mutant protein
palC114(1) −ts T43A,T44G F15S F15S
palC86(2) −ts T44C F15S F15S
palC103(2) −ts T61C S21P S21P
palC185(1) − A193insCGTTCTCTCCGCA I65fs 1–64 + 5
palC146(1) +/−− T194A,T195A I65K I65K
palC95(2) ++/− T202C Y68H Y68H
palC158(1) +/−− T202A Y68N Y68N
palC98(2) −ts G301A,T302G V101R V101R
palC111(1) − C306T,C307T Q103stop 1–102
palC155(1) −ts ΔT320–G322 L107Q,ΔE108 L107Q,ΔE108
palC93(2) −ts T331C W111R W111R
palC83(2) −ts T419A I140K I140K
palC127(1) − T438A Y146stop 1–145
palC134(1) − C461A S154stop 1–153
palC151(1) − ΔC461 S154stop 1–153
palC97(2) +/− T481G,A482T,T483G Y161V Y161V
palC99(2)a +/− T481G Y161D Y161D
palC91(2) +/− A566T H189L H189L
palC181(1) + T567G H189Q H189Q
palC100(2)a +/− T567A H189Q H189Q
palC149(1) − C570G,G571A S190stop 1–189
palC178(1) − C581G S194stop 1–193
palC141(1) − ΔA588–A600 P196fs 1–196 + 22
palC164(1)a +/− T653G,A654G L218R L218R
palC143(1) − T668G L223stop 1–222
palC129(1) − ΔA701 D234fs 1–233 + 51
palC82(2)a −ts G703A D235N D235N
palC81(1) −ts G703A,G718A D235N,A240T D235N,A240T
palC92(2) +/− A728C Q243P Q243P
palC148(1) − ΔT747 D249fs 1–248 + 36
palC150(1) − A748G,G755A,G756A K250E,W252stop K250E,1–251
palC85(2) +/− G755C W252S W252S
palC94(2) +/− G755C W252S W252S
palC84(2) −ts T813G C271W C271W
palC88(2) −ts A945T R315S R315S
palC80(1) − G961A G321D G321D
palC144(1) − ΔG984 G328fs 1–328 + 38
palC116(1) − ΔT1093 S365fs 1–364 + 2
palC160(1) − ΔC1114 R372fs 1–371 + 8
palC117(1) − A1135T K379stop 1–378
palC177(1) − G1159T E387stop 1–386
palC104(1) − T1181G L394stop 1–393
palC96(1)a − T1181A L394stop 1–393
palC89(2) + A1193T D398V D398V
palC112(1) − C1204T R402stop 1–401
palC153(1) − T1336G L427stop 1–426
palC179(1) − T1336A L427stop 1–426
palC159(1) − A1337insGA I428fs 1–427 + 29
palC90(2) +/− C1371T,C1372T P439F P439F
palC107(1) +/− C1371T,C1372T P439F P439F
palC162(1) − ΔG1378–C1380 ΔR442 ΔR442
palC113(1) +/− G1381A R442H R442H
palC87(2) +/−− T1407A Y451N Y451N
palC131(1) +/− T1419insTT S455fs 1–454 + 2
palC102(2) +/− T1423G L456R L456R
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Novel palC mutations were selected after UV mutagenesis of diploid R pabaA1 yA2 areAr18/biA1 areAr3 palC40 inoB2 fwA1 (see Clutterbuck 1993 for gene symbols) on minimal medium (Cove 1966) pH 6.5 with 1% glucose and 5 mm GABA. Mutants were screened for reduced growth on pH 8 medium (Cove 1976) to distinguish acidity-mimicking mutants. Acidity-mimicking mutant diploids were haploidized on benlate-containing Aspergillus complete medium (Cove 1966; Hastie 1970) lacking inositol and with NaH2PO4 added to 1 m. Haploid isolates were phenotype tested and categorized by their growth on pH 8 medium (Cove 1976), which is the most sensitive test to distinguish the relative leakiness of mutations. −, complete loss-of-function, virtually no growth at 25° or 37°; −ts, some growth at 25° but not at 37°; +/−−, +/−, and ++/−, increasing amounts of growth at 37°; +, similar to wild type on pH 8 medium yet acidity mimicking by more stringent criteria such as molybdate hypersensitivity (Caddick et al. 1986). All missense mutations except palC80 permitted significant growth at pH 8 at 25°. The subscript after the allele number refers to the experiment in which the mutation was isolated (see text). The difference in phenotype between the haploid palC181 (His189Gln) mutant and the aneuploid palC100 (His189Gln), “+” vs. “+/−,” respectively, suggests that the mutant phenotype of a new mutation in haploidy might be less pronounced than that in aneuploidy with palC40. fs, frameshift at the indicated codon; + N, where N is a number, indicates the number of frameshifted amino acid residues. Due to the large number of mutations selected, a relatively small number of strains were analyzed in some cases and the possibility cannot be ruled out that the phenotype might be affected by a modifying mutation, although an unexpected phenotype led to analysis in a cross. Naked DNA or DNA from conidiospores from haploid isolates was used as template for PCR amplification using palC-specific primers. Mutations were detected by the direct sequencing of PCR products. The entire coding region was sequenced for all new mutant alleles. Nucleotide numbers refer to the published sequence (Negrete-Urtasun et al. 1999).

a

The sequenced strain is probably aneuploid and carried palC40 in addition to the new palC mutation.

Figure 2.

Figure 2.

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Alignment of some ascomycete and basidiomycete PalCs and a zygomycete PalC. The alignment was carried out using the T-Coffee multiple sequence alignment (Notredame et al. 2000). Shading was according to the Blosum62 matrix: >90% similarity, solid background; 50–90% similarity, dark shading; 30–50% similarity, light shading. Residue-substituting mutations are indicated by ↑; different substitutions of the same residue are indicated by /; double mutations in the same allele are indicated by parentheses; ⇓ marks the ultimate wild-type residue in PalC159, the most C-terminal total loss-of-function truncated mutant protein; ↓ indicates the ultimate wild-type residue in the leaky truncated mutant PalC131 protein. Residues 38–235 composing the PCBROH domain (see Figure 4 and text) are italicized. A total of 73 Ustilago maydis PalC residues with no similarity to any other protein shown have been removed from the fifth block of the alignment. They are: 315-HAGTQIGLSANHEHELASRLSASRDRADHEHDDDMVETNRGAGAQATSKRNKLLGRFKLGSSKSSPPRSASVH-388. Initials refer to species names used in A. With the sole exception of the A. nidulans PalC, for which complete cDNA sequence is available, proteins from all other fungi were conceptually translated from predicted genes derived from genomic sequences. In Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, and U. maydis, for which automatic gene annotations were available, our deduced intron/exon organizations were coincident with automatic predictions (gene models NCU03316.1, FG05608.1, MG09311.4, and UM04392.1, respectively). See supplementary Table S1 at http://www.genetics.org/supplemental/ for sequence sources.

Figure 4.

Figure 4.

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Conserved regions in PalC. (A) The PalC BRO1 homology. Multiple sequence alignment of five ascomycete PalC proteins and representative members of each of five PF03097/BRO1-containing protein subfamilies illustrates sequence similarity between PalCs (indicated with a black bar on the left) and Bro1-like proteins. The five PF03097 subfamilies have been recognized by phylogenic analysis (J. C. Sánchez-Ferrero, O. Vincent and M. A. Peñalva, unpublished results) and comprise PalA ascomycete proteins (indicated with a red bar), BRO1 ascomycete proteins (yellow bar), metazoan AIP1(s)/Alix(es) (blue bar), rhophilins (RHPs, gray bar), and protein tyrosine phosphatases of the TD14 p164 class (HDPTPs, green bar). Fully (or nearly so) conserved residues are in magenta, with the consensus shown below the corresponding column. Blom62 similarity groups, where “6” indicates leucine, isoleucine, valine, and methionine, were used. Blue and yellow indicate decreasing degrees of conservation. Only columns having at least one residue conserved in both PalCs and Bro1s were shaded. Arrowheads denote PCBROH conserved residues substituted in extant loss-of-function mutants. An, A. nidulans; Ci, C. immitis; Fg, F. graminaerum; Nc, N. crassa; Cn, C. neoformans; Sc, S. cerevisiae; Hs, Homo sapiens; Ce, Caenorhabditis elegans; Fr, Fugu rubripes. See supplementary Tables S1 and S2 at http://www.genetics.org/supplemental/ for sources of PalC orthologous sequences and BRO1 domain-containing protein sequences, respectively. The PalC Hidden Markov model corresponding to A. nidulans residues 38–235 was constructed using HMMer 2.3.2 and 15 of the 17 available PalC ortholog sequences in the databases, including those in supplementary Table S1 at http://www.genetics.org/supplemental/ except the PalCs of Aspergillus fumigatus and Aspergillus oryzae, which were not included to avoid overrepresentation of sequences too closely related to the A. nidulans PalC. (B) Schematic of the PalC primary structure, where N-ter, LALA, RARA, and ERRE refer to blocks of strong identity/similarity as deduced from ascomycete alignment (see Figure 2); the C-terminal red oval is the fully conserved PalC di-aromatic motif (see Figure 2). Single residue mutant substitutions described in this work are indicated by red triangles (blue if more than one residue substitution is at that position). The PCBROH region is as defined in A. The black arrow indicates the limit of extant complete loss-of-function truncations, whereas the blue arrow indicates the position of a partial loss-of-function truncation.

PalC orthologs:

PalC orthologs are present in members of three major fungal phyla, ascomycota, basidiomycota, and zygomycota (Figures 2 and 3). Among ascomycete PalCs the Y. lipolytica ortholog Figure 2 is the first to be detectable in hemiascomycetes (yeasts). This dispels the notion, based on the failure to detect PalC homologs in the previously available hemiascomycete genomes (supplementary Table S1 at http://www.genetics.org/supplemental/) of Ashbya gosypii, C. albicans, Candida glabrata, Debaryomyces hansenii, Kluyveromyces lactis, S. cerevisiae, and Schizosaccharomyces pombe, that PalCs are exclusive to euascomycetes (filamentous fungi). As strong comparative genomic evidence indicates that Y. lipolytica separated early from the main hemiascomycete line and has not been subjected to the significant constraints in genome size characterizing other yeasts (Dujon et al. 2004), we suggest that palC was present in a universal fungal ancestor and that palC orthologs might have been lost from most hemiascomycetes.

Figure 3.

Figure 3.

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Phylogenetic tree relating PalC orthologs used in this work. This was constructed using MEGA version 3.0 neighbor-joining method (Kumar et al. 2004). D is a measure of sequence divergence.

Y. lipolytica palC is the only homolog of an A. nidulans pH regulatory gene not identified by mutations affecting pH regulation of extracellular proteases (Tréton et al. 2000; Gonzalez-Lopez et al. 2002). Thus its functional involvement in pH regulation remains to be established.

Refined similarity searching reveals the PalC-related protein family:

As sequence similarity searches gave no indication of a possible molecular role for PalC, we also used the relatively sensitive procedures of HMMer (Eddy 1998) and PSI-BLAST (Altschul et al. 1997) to seek PalC-related protein families. Searching the nrdbembl (January 2005) protein database with the 262 N-terminal residues of PalC detected, as well as PalC orthologs, we detected two members of the PFAM BRO1 domain family after the third round of iteration with E-values markedly below the cutoff (E = 0.005) and all 63 PFAM PF03097 BRO1-like domain-containing proteins after the eighth iteration and retrieved no new unrelated sequences subsequently. This relationship to BRO1 domain proteins was highly suggestive in view of the PalA BRO1 domain and was substantiated using a Hidden Markov model derived from the region of similarity corresponding to A. nidulans PalC 38–235 in the 14 PalCs available to query the nrdbembl, which detected three BRO1 domain proteins with reasonable scores (0.0018–0.046). In a third approach, a PalC Hidden Markov model in an HHpred (Söding 2005) search of the PFAM database gave only one significant hit (E-value 3e-05), the BRO1-like domain. These data provide statistical evidence that a region of PalC comprising residues 28–235 is significantly, albeit remotely, related to the PFAM PF03097 BRO1-like domain and a region of similarity extending downstream from it (Figure 4A). We have denoted this region PCBROH (PalC-BRO1 homology). The frequency of mutational changes here and their occurrence in key PCBROH residues such as Tyr68 and Trp111 indicate a major functional and/or structural role in PalC (Figure 4B).

BRO1 domain proteins include, as well as PalA (Negrete-Urtasun et al. 1997), PalA orthologs Rim20p (S. cerevisiae and C. albicans) (Xu and Mitchell 2001), the human PalA homolog AIP1/Alix (Missotten et al. 1999; Vito et al. 1999), and the yeast Bro1p (Nickas and Yaffe 1996), an MVB pathway protein recruiting the deubiquitinase Doa4p to endosomes (Odorrizi et al. 2003; Luhtala and Odorrizi 2004). All these proteins interact with Vps32p/Snf7p (a key component of ESCRT-III) or its homologs (Xu and Mitchell 2001; Katoh et al. 2003; Strack et al. 2003; Vincent et al. 2003; von Schwedler et al. 2003; Katoh et al. 2004; Peck et al. 2004; Xu et al. 2004).

It has been suggested that the BRO1 domain is the region of interaction between Rim20p-Bro1p family members and Snf7p family members (Xu et al. 2004). However, the finding that the BRO1 domain-containing protein human rhophilin-2 did not interact with either of two human hSnf7 proteins tested (Peck et al. 2004) argues against this generalization. Thus a precise role for the BRO1-like domain in PalC cannot be assigned. However, it is tempting to speculate that it reflects a further link between the pH response system and the MVB pathway multiprotein complexes at the endosome and/or cell membrane.

The PacC-mediated pH regulatory system is an important virulence determinant in plant (reviewed by Peñalva and Arst 2004) and animal pathogens, including C. albicans (reviewed by Fonzi 2002; Peñalva and Arst 2002; Davis 2003) and A. nidulans (Bignell et al. 2005). In view of the growing frequency of invasive mold, and in particular of Aspergillus infections (reviewed by Clark and Hajjeh 2002), the uniquely fungal PalC might possibly be a suitable target for therapeutic intervention.

Acknowledgments

We are grateful to Lily Stanton for technical assistance; Elaine Bignell, Eduardo Espeso, and Olivier Vincent for comments on the manuscript; and Joanna Rudnicka for interesting discussion. We thank the Wellcome Trust, the Dirección General de Investigación Científica y Técnica, and the Consejo Superior de Investigaciones Científicas (CSIC) for their support through grants 067878, BIO2003-0077, and for a CSIC Bioinformatics I3P studentship for J.C.S.-F., respectively.

Note added in proof: The structure of the Bro1 domain in yeast Bro1p has been determined (J. Kim, S. Sitaraman, A. Hierro, B. M. Beach, G. Odorrizi and J. H. Hurley, 2005, Structural basis for endosomal targeting by the Bro1 domain. Dev. Cell 8: 937–947) and establishes that the Bro1 domain contains 367 residues, thereby extending considerably beyond the 160-residue Bro1p PFAM domain. Sequence alignment suggests that the region of A. nidulans PalC corresponding to the structurally based Bro1 domain of Bro1p includes the PalC N-terminal 446 residues, within which the majority of our single residue substitutions fall. Conserved PalC residues Arg47, Tyr68, Trp111, and Glu136 (Figures 2 and 4A) are likely to correspond, respectively, to Bro1p residues Arg51, Tyr70, Trp94, and Glu116, which are central in one of two structure-stabilizing, buried, charged, and polar clusters. The loss-of-function phenotypes of palC Tyr68His or -Asn and Trp111Arg mutations are consistent with the predicted structural importance of these residues.

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