Mutational analysis of the pH signal transduction component PalC of Aspergillus nidulans supports distant similarity to BRO1 domain family members

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.

Figure 1.

Diploid R (used for palC mutant selection). (A) Chromosomes III and IV of diploid R areA^r18/areA^r3 palC40 inoB2. The diploid was constructed using standard classical genetic techniques (Clutterbuck 1993) between parents of the relevant partial genotypes areA^r18 (nitrogen metabolite repressed) (Arst et al. 1989) and inoB2 (inositol requiring), areA^r3 (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 areA^r18 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 areA^r18 breakpoint and palC40 and a second between palC40 and inoB2, inoB⁺ palC40 homozygotes can result.

Figure 2.

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 3.

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.

Figure 4.

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.