Article Figures & Data
Figures
- 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.
MYC3-tagging pacC by gene replacement. Nc., Neurospora crassa.
- 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.
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.
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.
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.
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.
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
- TABLE 1.
MYC3-tagged pacC alleles and strains constructed and characterized in this worka
Allele Change(s) in protein Nucleotide change(s) Strain name Genotype Result by Western analysis pacC900(L481A) L481A C2399G T2400C MP87 pacC900(L481A) + pacC900(G482A) G482A G2403C MP123 pacC900(G482A) − pacC900(F485A) F485A T2411G T2412C MP91 yA2 pacC900(F485A) + pacC900(D486A) D486A A2415C MP114 yA2 pabaA1 pacC900(D486A) + pacC900(D488A) D488A A2421C MP84 yA2 inoB2 pacC900(D488A) + pacC900(E489A) E489A A2424C MP160 yA2 pabaA1 inoB2 pacC900(E489A) + pacC900(R490A) R490A C2426G G2427C MP82 pacC900(R490A) − pacC900(R491A) R491A C2429G G2430C MP79 pacC900(R491A) + pacC900(R491A Q499A) R491A C2429G G2430C MP116 inoB2 pacC900(R491A Q499A) + Q499A C2453G A2454C pacC900(R492A) R492A A2432G G2433C MP121 yA2 pabaA1 pacC900(R492A) − pacC900(Y493A) Y493A T2435G A2436C MP104 inoB2 pacC900(Y493A) + pacC900(T494A) T494A A2438G MP94 yA2 pabaA1 pacC900(T494A) + pacC900(G495A) G495A G2442C MP100 pacC900(G495A) + pacC900(G496A) G496A G2445C MP98 pacC900(G496A) − pacC900(L498A) L498A T2450G MP190 adE20 pyroA4 pacC900(L498A) + pacC900(L498F) L498F G2452C MP212 adE20 pacC900(L498F) + pacC900(L498S) L498S T2451C MP176 adE20 pacC900(L498S) + pacC900(Q499A) Q499A C2453G A2454C MP76 inoB2 pacC900(Q499A) + pacC900 None None yA2 pabaA1 pacC900 + ↵ a Amino acids and nucleotides are as described by Tilburn et al. (69).
- TABLE 2.
palB mutations characterized in this worka
Allele Nucleotide change Change in protein Growth at pH 8.0d Mutagen method Selection Reference 20°C 37°C Wild type None None +++++ +++++ None None 8, 67 Truncating mutations palB508 T340G L13stop − − UV Diploid GABA This work palB510 T340A L13stop − − UV Diploid GABA This work palB70 A359T PSD ND − None Haploid GABA E. A. Espeso (unpublished) palB38 A515T K55stop − − UV Haploid GABA This work palB509 del(658-662) R103fsc − − UV Diploid GABA This work palB502 T678G L109stop − − UV Diploid GABA This work palB501 C680T Q110stop − − UV Diploid GABA This work palB518 T849C PSD ++ +/− UV Diploid GABA This work palB520 A866T K144stop − − UV Diploid GABA This work palB519 A1638T K385stop − − UV Diploid GABA This work palB500 G1767T E428stop − − UV Diploid GABA This work palB505 delA2007 K508fs − − UV Diploid GABA This work palB513 T2140G L552stop − − UV Diploid GABA This work palB511 delG2801 A2803G Y773fs − − UV Diploid GABA This work palB111 G2784T E767stop − − NTG Haploid GABA This work palB7 G2856T G791stop +/− − UV Lack of alkaline phosphatase 18 palB11b del(G2863-C2873) R793fs − − UV Lack of alkaline phosphatase 18 palB37 G2925T E814stop +/− − UV Haploid GABA This work Missense mutations palB517 T1027G G1029T D197E C198F − − UV Diploid GABA This work palB16 GT(1034 1035)AA V200N − − UV Haploid GABA This work palB504 T1405G L307R + − UV Diploid GABA This work palB524 T2388A I618L ++++ +++ UV Diploid GABA This work palB39 T2340C S619P +++ − UV Haploid GABA This work palB515 T2713C L743P ++ − UV Diploid GABA This work palB516 A2778C T2783C T765P ++ − UV Diploid GABA This work palB503 T2875C F797S +++ +++ UV Diploid GABA This work palB514 T2923C L813P ++ − UV Diploid GABA This 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.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Oligonucleotides used in the mutagenesis of the signaling protease box.
MS Word document, 28K.
- Supplemental file 1
-
Oligonucleotides used in the mutagenesis of the signaling protease box.
