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Further Characterization of the Signaling Proteolysis Step in the Aspergillus nidulans pH Signal Transduction Pathway

María M. Peñas, América Hervás-Aguilar, Tatiana Múnera-Huertas, Elena Reoyo, Miguel Á. Peñalva, Herbert N. Arst Jr., Joan Tilburn
María M. Peñas
1Department of Molecular Microbiology and Infection, Imperial College London, The Flowers Building, London SW7 2AZ, United Kingdom
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América Hervás-Aguilar
2Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, Madrid 28040, Spain
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Tatiana Múnera-Huertas
1Department of Molecular Microbiology and Infection, Imperial College London, The Flowers Building, London SW7 2AZ, United Kingdom
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Elena Reoyo
2Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, Madrid 28040, Spain
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Miguel Á. Peñalva
2Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, Madrid 28040, Spain
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Herbert N. Arst Jr.
1Department of Molecular Microbiology and Infection, Imperial College London, The Flowers Building, London SW7 2AZ, United Kingdom
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Joan Tilburn
1Department of Molecular Microbiology and Infection, Imperial College London, The Flowers Building, London SW7 2AZ, United Kingdom
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  • For correspondence: j.tilburn@imperial.ac.uk
DOI: 10.1128/EC.00047-07
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  • FIG. 1.
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    FIG. 1.

    Model of PacC two-step processing. Codon 5 is the physiological pacC initiation codon (42). It is not known if PalA and PalC interact with Vps32 simultaneously or sequentially.

  • FIG. 2.
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    FIG. 2.

    MYC3-tagging pacC by gene replacement. Nc., Neurospora crassa.

  • FIG. 3.
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    FIG. 3.

    Sequence features of PalB and palB mutations. Recognized domains within PalB are schematized. MIT, microtubule interacting and trafficking domain (11, 59, 61, 71); Calpain_II, calpain domain II; PalBh, PalB homologous domain; Calpain_III, Calpain large subunit domain III (16, 25, 26, 32, 57, 65, 66; for a review, see reference 63). Stars highlight the positions of the catalytic triad residues, cysteine, histidine, and asparagine/aspartate. The conserved regions around the catalytic triad residues, cysteine (residues 191 to 202) and histidine and asparagine/aspartate (residues 361 to 388), and conserved and rather poorly conserved regions around Leu307Arg (residues 300 to 313) and Ile618Leu and Ser619Pro (612 to 622), respectively, are shown above the scheme. Missense and truncating mutations in the C terminus (residues 700 to 847) are shown below the scheme. Residue changes and truncating mutations are indicated with upward- and downward-pointing arrows, respectively. Null mutations are indicated with solid-headed arrows, and those retaining some function are indicated with open-headed arrows. Abbreviations and accession numbers (emb, EMBL; sp, SwissProt; up, UniProt) are as follows: An, A. nidulans, emb Z54244; Ci, Coccidioides immitis, up Q1DXZ5; Hc, Histoplasma capsulatum, retranslated from sequence from locus HCAG_04795.1 (http://www.broad.mit.edu/annotation/genome/histoplasma_capsulatum/ ); Chg, Chaetomium globosum, up Q2H9W2; Nc, Neurospora crassa, sp Q7RZP7; Gz, Gibberella zeae, up Q4IBM8; Phn, Phaesophaeria nodorum, up Q0U7L9; Cn, Cryptococcus neoformans, sp Q55IT8; Um, Ustilago maydis, up Q4PCT8; Yl, Yarrowia lipolytica, sp Q9HFC8; Ca, Candida albicans, sp Q5AK25; Dh, Debaromyces hansenii, Q6BH66; Ag, Ashbya gossipii, Q759K3; Kl, Kluyveromyces lactis, sp Q6CKY3; Cg, Candida glabrata, Q6FJ28; Sc, S. cerevisiae, Q03792; m-c, human m-calpain, sp Q17655. Residues removed from the C. globosum and N. crassa sequences are (−25) VGRGGGGSGGESSGGGGGGSAVRVSLEVG and (−49) IEGRKRVLASTASGGGGELAASLSNLSLSERLGGIGGIGGGHIHG, respectively, as indicated. Shading is according to Blosom 62 similarity groups (DN, EQ, KR, ST, ILMV, and FYW): 50% similar, black; 40% similar, dark gray; 30% similar, light gray.

  • FIG. 4.
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    FIG. 4.

    PalB protein levels are not pH regulated. Western blot analysis of HA3-tagged PalB in palF+ and palF15 cells which were grown in acidic medium (H+) and harvested, indicated by “0” above the lane, or shifted to acidic or alkaline (OH−) medium for 15 or 30 min, as indicated above the lane. Actin loading controls were obtained from a Western blot of a duplicate gel.

  • FIG. 5.
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    FIG. 5.

    PalB is required for signaling proteolysis but not processing proteolysis of PacC. Western blot analysis of MYC3-tagged PacC processing in palB+ and palB38 strains (A) grown under acidic conditions and shifted to alkaline conditions for the times indicated above the panel or (B) grown under steady-state acidic or alkaline conditions, as indicated, with MYC3-tagged PacCc14 (Y493stop), where PacC is mutationally truncated within the signaling protease box. There is some heterogeneity at the C terminus of PacC27 (42).

  • FIG. 6.
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    FIG. 6.

    Conservation of the PacC signaling protease box. Interspecies alignment shows conserved residues in the region of the 24-residue signaling protease box described by Díez et al. (17). Identical or similar (Ser/Thr, Iso/Val, Asp/Glu, Phe/Tyr, and Arg/Lys) residues are highlighted in dark or light gray, respectively. Abbreviations and accession numbers (Chg, Chaetomium globosum database at http://www.broad.mit.edu/annotation/genome/chaetomium_globosum/ ; dbj, DNA Data Base Japan; emb, EMBL; gb, GenBank; sp, SwissProt; up, UniPro) are as follows: An, A. nidulans, emb Z47081.1; Ap, Aspergillus parasiticum, gb AAK98616.1; Ao, Aspergillus oryzae, dbj BAE57899.1; Ang, Aspergillus niger, emb CAA67063; Pc, Penicillium chrysogenum, gb AAC36492.1; Ag, Aspergillus giganteus, gb AAV28549.1; Af, Aspergillus fumigatus, sp Q4WY67; Ci, Coccidioides immitis, gb EAS32701.1; Tr, Trichophyton rubrum, gb AAK35072.2; Hc, Histoplasma capsulatum, sp Q52B93; Ss, Sclerotinia sclerotium, sp Q9P413; Bf, Botryotinia fuckeliana, gb AAV54519.1; Ac, Acremonium chrysogenum, spQ96X49; Fo, Fusarium oxysporum, sp Q870A3; Gm, Gibberella moniformis, sp Q873X0; Gf, Gibberella fujikuroi, sp Q8J1U9; Pn, Phaeosphaeria nodorum, gb EAT80783.1; Nc, Neurospora crassa, sp Q7RVQ8; Mg, Magnaportha grisea, sp Q52B93; Cg, Chaetomium globosum, Chg CHGG_05804.1. A. nidulans PacC amino acid numbers (69) and consequences of the pacCΔ13 deletion and classically derived pacC+/−209 and pacC+/−210 mutations (17) are shown above the alignment. Residues mutated in MYC-tagged PacC in this work are presented below the alignment.

  • FIG. 7.
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    FIG. 7.

    Phenotype testing of signaling protease box mutants in plate tests. The wild-type strain in row 1 is an untagged biA1 (biotin-requiring) strain. The difference in colony color on synthetic complete or neomycin-containing medium is due to differently pigmented (green or yellow) conidia and has no effect on pH characteristics. Acid phosphatase staining was carried out after growth on pH-8 minus-phosphate medium.

  • FIG. 8.
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    FIG. 8.

    Western blot analysis of PacC forms in signaling protease box mutants in response to shifting from acidic to alkaline growth medium. The positions of the PacC forms are indicated on the left.

Tables

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  • TABLE 1.

    MYC3-tagged pacC alleles and strains constructed and characterized in this worka

    AlleleChange(s) in proteinNucleotide change(s)Strain nameGenotypeResult by Western analysis
    pacC900(L481A)L481AC2399G T2400CMP87 pacC900(L481A)+
    pacC900(G482A)G482AG2403CMP123 pacC900(G482A)−
    pacC900(F485A)F485AT2411G T2412CMP91 yA2 pacC900(F485A)+
    pacC900(D486A)D486AA2415CMP114 yA2 pabaA1 pacC900(D486A)+
    pacC900(D488A)D488AA2421CMP84 yA2 inoB2 pacC900(D488A)+
    pacC900(E489A)E489AA2424CMP160 yA2 pabaA1 inoB2 pacC900(E489A)+
    pacC900(R490A)R490AC2426G G2427CMP82 pacC900(R490A)−
    pacC900(R491A)R491AC2429G G2430CMP79 pacC900(R491A)+
    pacC900(R491A Q499A)R491AC2429G G2430CMP116 inoB2 pacC900(R491A Q499A)+
    Q499AC2453G A2454C
    pacC900(R492A)R492AA2432G G2433CMP121 yA2 pabaA1 pacC900(R492A)−
    pacC900(Y493A)Y493AT2435G A2436CMP104 inoB2 pacC900(Y493A)+
    pacC900(T494A)T494AA2438GMP94 yA2 pabaA1 pacC900(T494A)+
    pacC900(G495A)G495AG2442CMP100 pacC900(G495A)+
    pacC900(G496A)G496AG2445CMP98 pacC900(G496A)−
    pacC900(L498A)L498AT2450GMP190 adE20 pyroA4 pacC900(L498A)+
    pacC900(L498F)L498FG2452CMP212 adE20 pacC900(L498F)+
    pacC900(L498S)L498ST2451CMP176 adE20 pacC900(L498S)+
    pacC900(Q499A)Q499AC2453G A2454CMP76 inoB2 pacC900(Q499A)+
    pacC900 NoneNone yA2 pabaA1 pacC900 +
    • ↵ a Amino acids and nucleotides are as described by Tilburn et al. (69).

  • TABLE 2.

    palB mutations characterized in this worka

    AlleleNucleotide changeChange in proteinGrowth at pH 8.0dMutagen methodSelectionReference
    20°C37°C
    Wild typeNoneNone++++++++++NoneNone 8, 67
    Truncating mutations
        palB508T340GL13stop−−UVDiploid GABAThis work
        palB510T340AL13stop−−UVDiploid GABAThis work
        palB70A359TPSDND−NoneHaploid GABAE. A. Espeso (unpublished)
        palB38A515TK55stop−−UVHaploid GABAThis work
        palB509del(658-662)R103fsc−−UVDiploid GABAThis work
        palB502T678GL109stop−−UVDiploid GABAThis work
        palB501C680TQ110stop−−UVDiploid GABAThis work
        palB518T849CPSD+++/−UVDiploid GABAThis work
        palB520A866TK144stop−−UVDiploid GABAThis work
        palB519A1638TK385stop−−UVDiploid GABAThis work
        palB500G1767TE428stop−−UVDiploid GABAThis work
        palB505delA2007K508fs−−UVDiploid GABAThis work
        palB513T2140GL552stop−−UVDiploid GABAThis work
        palB511delG2801 A2803GY773fs−−UVDiploid GABAThis work
        palB111G2784TE767stop−−NTGHaploid GABAThis work
        palB7G2856TG791stop+/−−UVLack of alkaline phosphatase 18
        palB11bdel(G2863-C2873)R793fs−−UVLack of alkaline phosphatase 18
        palB37G2925TE814stop+/−−UVHaploid GABAThis work
    Missense mutations
        palB517T1027G G1029TD197E C198F−−UVDiploid GABAThis work
        palB16GT(1034 1035)AAV200N−−UVHaploid GABAThis work
        palB504T1405GL307R+−UVDiploid GABAThis work
        palB524T2388AI618L+++++++UVDiploid GABAThis work
        palB39T2340CS619P+++−UVHaploid GABAThis work
        palB515T2713CL743P++−UVDiploid GABAThis work
        palB516A2778C T2783CT765P++−UVDiploid GABAThis work
        palB503T2875CF797S++++++UVDiploid GABAThis work
        palB514T2923CL813P++−UVDiploid GABAThis work
    • ↵ a Nucleotides and amino acids are numbered as in GenBank Z54244. PSD, predicted splicing defect; ND, not determined; NTG, N-methyl-N′-nitro-N-nitrosoguanidine.

    • ↵ b Formerly palE11; see the work of Arst et al. (1). The palB11 phenotype, which is more extreme than that of the more N-terminal nonsense palB7 mutation, is possibly influenced by 17 out-of-frame residues at the C terminus of the protein.

    • ↵ c fs, frameshift.

    • ↵ d −, no growth; +/−, very small amount of growth; + to +++++, increasing amounts of growth from small to wild-type.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Oligonucleotides used in the mutagenesis of the signaling protease box.
      MS Word document, 28K.
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Further Characterization of the Signaling Proteolysis Step in the Aspergillus nidulans pH Signal Transduction Pathway
María M. Peñas, América Hervás-Aguilar, Tatiana Múnera-Huertas, Elena Reoyo, Miguel Á. Peñalva, Herbert N. Arst Jr., Joan Tilburn
Eukaryotic Cell Jun 2007, 6 (6) 960-970; DOI: 10.1128/EC.00047-07

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Further Characterization of the Signaling Proteolysis Step in the Aspergillus nidulans pH Signal Transduction Pathway
María M. Peñas, América Hervás-Aguilar, Tatiana Múnera-Huertas, Elena Reoyo, Miguel Á. Peñalva, Herbert N. Arst Jr., Joan Tilburn
Eukaryotic Cell Jun 2007, 6 (6) 960-970; DOI: 10.1128/EC.00047-07
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KEYWORDS

Aspergillus nidulans
Cysteine Endopeptidases
Fungal Proteins
Hydrogen-Ion Concentration
Signal Transduction
Transcription Factors

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