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

By Tilburn, Joan; Sánchez-Ferrero, Juan C. et al. | Genetics, September 2005 | Go to article overview

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


Tilburn, Joan, Sánchez-Ferrero, Juan C., Reoyo, Elena, Arst, Herbert N., Jr., A, Miguel, Genetics


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 PENALVA and ARST 2002, 2004; ARST and PENALVA 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 (UREJAS et al. 1995; MINGOT et al. 1999; DÎEZ 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 alkalineexpressed genes (ESPESO and PENALVA 1996) and repressor of acid-expressed genes (ESPESO and ARST 2000).

Signal transduction components PaIH and Pall (S. cerevisiaehomologs Rim21p and Rim9p, respectively) are predicted seven- and four-pass membrane proteins (Li and MITCHELL 1997; DENISON et al. 1998; NEGRETEURTASUN et al. 1999) and strong candidates as ambient pH sensors. PaIB (S. cerevisiae Riml3p), 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. PaIA (S. cerevisiae Rim20p) (NEGRETE-URTASUN et al. 1997; Xu and MITCHELL 2001) contains the ^160-residue BROl domain (PFAM domain PF03097; httpr/^www-sanger. ac.uk/Software/Pfam/index.shtml), first identified in yeast Brolp (NiCKAS and YAFFE 1996). PaIA 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 mdosomal sorting complex required for iransport-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 (PaIA) interacts with both RimlOlp (PacC) (Xu and MITCHELL 2001) and Vsp32p/Snf7p, which also interacts with Riml3p (PaIB) (Éôï et al. 2001), to form a scaffold-promoting interaction between the RimlOlp cleavage site and the Riml3p 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 PaIF (S. cerevisiaeRimSp) and PaIC, which has no S. cerevisiae homolog (Li and MITCHELL 1997; MACCHERONI et al. 1997; NEGRETE-URTASUN et al. 1999). As the PaIC primary amino acid sequence revealed no evident sequence signature and PaIC appeared absent from the hemiascomycete lineage, we carried out rnutational analysis of this protein along with sequence profile similarity searching, exploiting the recent publication of a number of fungal genomes.

Mutational analysis: The GABA (-/-aminobutyrate) technique is a powerful tool for the selection of pacC (MiNGOT et al. …

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