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PalC, one of two Bro1 domain proteins in the fungal pH signalling pathway, localizes to cortical structures and binds Vps32.

Galindo A, Hervás-Aguilar A, Rodríguez-Galán O, Vincent O, Arst HN, Tilburn J, Peñalva MA - Traffic (2007)

Bottom Line: Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active.This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence.A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain.

ABSTRACT
PalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Delta impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

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Single-residue substitutions/deletion within PalC conserved regions I and II largely impair or prevent its interaction with Vps32.A) Western blot analysis of yeast strains expressing the indicated fusion proteins, which were used as preys in two-hybrid assays. Fusion proteins were detected with anti-HA antibody. Actin was used as a loading control. B) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quadruple selection on -Leu, -Trp, -His, -Ade medium using strain AH109. The experiment was carried out in triplicate. QSM indicates quadruple selection medium. -W-L is medium lacking Tp and Leu. C) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quantitative β-galactosidase assays using strain Y187. Bars indicate standard errors.
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fig04: Single-residue substitutions/deletion within PalC conserved regions I and II largely impair or prevent its interaction with Vps32.A) Western blot analysis of yeast strains expressing the indicated fusion proteins, which were used as preys in two-hybrid assays. Fusion proteins were detected with anti-HA antibody. Actin was used as a loading control. B) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quadruple selection on -Leu, -Trp, -His, -Ade medium using strain AH109. The experiment was carried out in triplicate. QSM indicates quadruple selection medium. -W-L is medium lacking Tp and Leu. C) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quantitative β-galactosidase assays using strain Y187. Bars indicate standard errors.

Mentions: We mapped the region of PalC involved in binding Vps32 by testing the effects of PalC single-residue substitutions in two-hybrid assays. While Arg442Δ is phenotypically in vivo, PalC substitutions Tyr451Asn, Pro439Phe and Arg442His (Figure 2) lead to considerable yet slightly incomplete loss of function, as determined using the most sensitive tests (34). In an attempt to exacerbate the phenotype of substitutions involving Arg442 and Tyr451, we constructed Arg442Ala and Tyr451Ala, which were confirmed as leading to loss of function (see below). A panel of single-residue substitutions (Pro439Phe, Arg442His, Arg442Ala and Tyr451Ala), a single-residue deletion (Arg442Δ) and two truncating mutations (after residues 426 and 454) were introduced in the GAL4AD::PalC protein fusion and tested against the A. nidulansVps32 bait. Western blot analysis of S. cerevisiaestrains expressing mutant preys revealed that, with the exception of the truncating mutations, whose steady-state level was reduced relative to wild type (and were thus not considered any further), all other mutations did not markedly affect fusion protein levels (Figure 4A), suggesting that these do not result in major adverse effects in PalC folding. Growth tests on selective media (Figure 4B) and β-galactosidase assays (Figure 4C) showed that PalC single-residue mutations largely impaired (Arg442His and Arg442Ala) or prevented (Pro439Phe, Tyr451Ala and Arg442Δ) the two-hybrid interaction. Of note, Arg442Ala and Arg442His reduced β-galactosidase activity to ∼18 and ∼11% of the wild type, respectively, which contrasts with the total lack of activity using Arg442Δ, despite the similar steady-state levels of these three mutant PalC preys. The partial and complete loss of function phenotypes of Arg442His and Arg442Δ, respectively, in two-hybrid assays correlate with the phenotypic effects of the corresponding mutant alleles in vivo(34) (see above). These data strongly support the above conclusion that the PalC–Vps32 interaction is specific and point to the conserved regions containing Arg442, Pro439 and possibly Tyr451 (Figure 2) as likely candidate(s) that mediate, at least in part, this specificity. The Bro1 region corresponding to the environs of PalC Tyr451 is not included in the crystal structure. However, as discussed above, Pro439 and Arg442 are within a region that in Bro1p is involved in Vps32p binding (Figure 2).


PalC, one of two Bro1 domain proteins in the fungal pH signalling pathway, localizes to cortical structures and binds Vps32.

Galindo A, Hervás-Aguilar A, Rodríguez-Galán O, Vincent O, Arst HN, Tilburn J, Peñalva MA - Traffic (2007)

Single-residue substitutions/deletion within PalC conserved regions I and II largely impair or prevent its interaction with Vps32.A) Western blot analysis of yeast strains expressing the indicated fusion proteins, which were used as preys in two-hybrid assays. Fusion proteins were detected with anti-HA antibody. Actin was used as a loading control. B) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quadruple selection on -Leu, -Trp, -His, -Ade medium using strain AH109. The experiment was carried out in triplicate. QSM indicates quadruple selection medium. -W-L is medium lacking Tp and Leu. C) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quantitative β-galactosidase assays using strain Y187. Bars indicate standard errors.
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Related In: Results  -  Collection

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fig04: Single-residue substitutions/deletion within PalC conserved regions I and II largely impair or prevent its interaction with Vps32.A) Western blot analysis of yeast strains expressing the indicated fusion proteins, which were used as preys in two-hybrid assays. Fusion proteins were detected with anti-HA antibody. Actin was used as a loading control. B) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quadruple selection on -Leu, -Trp, -His, -Ade medium using strain AH109. The experiment was carried out in triplicate. QSM indicates quadruple selection medium. -W-L is medium lacking Tp and Leu. C) PalC mutations affecting two-hybrid interaction with Vps32, as determined by quantitative β-galactosidase assays using strain Y187. Bars indicate standard errors.
Mentions: We mapped the region of PalC involved in binding Vps32 by testing the effects of PalC single-residue substitutions in two-hybrid assays. While Arg442Δ is phenotypically in vivo, PalC substitutions Tyr451Asn, Pro439Phe and Arg442His (Figure 2) lead to considerable yet slightly incomplete loss of function, as determined using the most sensitive tests (34). In an attempt to exacerbate the phenotype of substitutions involving Arg442 and Tyr451, we constructed Arg442Ala and Tyr451Ala, which were confirmed as leading to loss of function (see below). A panel of single-residue substitutions (Pro439Phe, Arg442His, Arg442Ala and Tyr451Ala), a single-residue deletion (Arg442Δ) and two truncating mutations (after residues 426 and 454) were introduced in the GAL4AD::PalC protein fusion and tested against the A. nidulansVps32 bait. Western blot analysis of S. cerevisiaestrains expressing mutant preys revealed that, with the exception of the truncating mutations, whose steady-state level was reduced relative to wild type (and were thus not considered any further), all other mutations did not markedly affect fusion protein levels (Figure 4A), suggesting that these do not result in major adverse effects in PalC folding. Growth tests on selective media (Figure 4B) and β-galactosidase assays (Figure 4C) showed that PalC single-residue mutations largely impaired (Arg442His and Arg442Ala) or prevented (Pro439Phe, Tyr451Ala and Arg442Δ) the two-hybrid interaction. Of note, Arg442Ala and Arg442His reduced β-galactosidase activity to ∼18 and ∼11% of the wild type, respectively, which contrasts with the total lack of activity using Arg442Δ, despite the similar steady-state levels of these three mutant PalC preys. The partial and complete loss of function phenotypes of Arg442His and Arg442Δ, respectively, in two-hybrid assays correlate with the phenotypic effects of the corresponding mutant alleles in vivo(34) (see above). These data strongly support the above conclusion that the PalC–Vps32 interaction is specific and point to the conserved regions containing Arg442, Pro439 and possibly Tyr451 (Figure 2) as likely candidate(s) that mediate, at least in part, this specificity. The Bro1 region corresponding to the environs of PalC Tyr451 is not included in the crystal structure. However, as discussed above, Pro439 and Arg442 are within a region that in Bro1p is involved in Vps32p binding (Figure 2).

Bottom Line: Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active.This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence.A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain.

ABSTRACT
PalC, distantly related to Saccharomyces cerevisiae peripheral endosomal sorting complexes required for transport III (ESCRT-III) component Bro1p and one of six Aspergillus nidulans pH signalling proteins, contains a Bro1 domain. Green fluorescent protein (GFP)-tagged PalC is recruited to plasma membrane-associated punctate structures upon alkalinization, when pH signalling is active. PalC recruitment to these structures is dependent on the seven transmembrane domain (7-TMD) receptor and likely pH sensor PalH. PalC is a two-hybrid interactor of the ESCRT-III Vps20/Vps32 subcomplex and binds Vps32 directly. This binding is largely impaired by Pro439Phe, Arg442Ala and Arg442His substitutions in a conserved region mediating interaction of Bro1p with Vps32p, but these substitutions do not prevent cortical punctate localization, indicating Vps32 independence. In contrast, Arg442Delta impairs Vps32 binding and prevents PalC-GFP recruitment to cortical structures. pH signalling involves a plasma membrane complex including the 7-TMD receptor PalH and the arrestin-like PalF and an endosomal membrane complex involving the PalB protease, the transcription factor PacC and the Vps32 binding, Bro1-domain-containing protein PalA. PalC, which localizes to cortical structures and can additionally bind a component of ESCRT-III, has the features required to bridge these two entities. A likely S. cerevisiae orthologue of PalC has been identified, providing the basis for a unifying hypothesis of gene regulation by ambient pH in ascomycetes.

Show MeSH
Related in: MedlinePlus