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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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Arg442Δ but not other mutations impairing or preventing PalC interaction with Vps32 prevents the alkaline pH-dependent increase in punctate subcellular localization of PalC-GFP.A) Western blot analysis of mutant PalC-GFP proteins. Mycelia of strains carrying wild type or mutant alcAp::PalC-GFP transgenes, as indicated, were precultured under derepressing, noninduced conditions and shifted to repressing (R) or inducing (I) conditions before proceeding to protein extraction and Western blot analyses using anti-GFP antibodies or anti-actin as loading control. B) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH as in legend to Figure 6. C and D) Representative images of PalC-GFP localization of wild type and mutants under alkaline conditions. Bars, 5 μm.
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fig07: Arg442Δ but not other mutations impairing or preventing PalC interaction with Vps32 prevents the alkaline pH-dependent increase in punctate subcellular localization of PalC-GFP.A) Western blot analysis of mutant PalC-GFP proteins. Mycelia of strains carrying wild type or mutant alcAp::PalC-GFP transgenes, as indicated, were precultured under derepressing, noninduced conditions and shifted to repressing (R) or inducing (I) conditions before proceeding to protein extraction and Western blot analyses using anti-GFP antibodies or anti-actin as loading control. B) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH as in legend to Figure 6. C and D) Representative images of PalC-GFP localization of wild type and mutants under alkaline conditions. Bars, 5 μm.

Mentions: We introduced, in the alcAp::palC-GFPtransgene, mutations leading to PalC Arg442Ala, Pro439Phe or Tyr451Ala substitutions or to the Arg442Δ in-frame deletion. None of these mutant transgenes complemented a phenotypically palC4allele in plate tests, which demonstrates that Arg442Ala and Tyr451Ala substitutions result in a loss of function phenotype (Figure S2). Western blot analyses showed that, under inducing conditions, Arg442Ala and Arg442Δ mutant transgenes synthesized full-length PalC-GFP proteins with steady-state levels similar to the wild type (Figure 7A, compare anti-GFP lanes 4 and 6 with lane 2). In contrast, Tyr451Ala and Pro439Phe PalC-GFP levels were lower (Figure 7A, lanes 8 and 10). Detection of degradation bands in the Tyr451Ala mutant indicates that this particular sequence change leads to decreased protein stability in Aspergillus.


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)

Arg442Δ but not other mutations impairing or preventing PalC interaction with Vps32 prevents the alkaline pH-dependent increase in punctate subcellular localization of PalC-GFP.A) Western blot analysis of mutant PalC-GFP proteins. Mycelia of strains carrying wild type or mutant alcAp::PalC-GFP transgenes, as indicated, were precultured under derepressing, noninduced conditions and shifted to repressing (R) or inducing (I) conditions before proceeding to protein extraction and Western blot analyses using anti-GFP antibodies or anti-actin as loading control. B) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH as in legend to Figure 6. C and D) Representative images of PalC-GFP localization of wild type and mutants under alkaline conditions. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171039&req=5

fig07: Arg442Δ but not other mutations impairing or preventing PalC interaction with Vps32 prevents the alkaline pH-dependent increase in punctate subcellular localization of PalC-GFP.A) Western blot analysis of mutant PalC-GFP proteins. Mycelia of strains carrying wild type or mutant alcAp::PalC-GFP transgenes, as indicated, were precultured under derepressing, noninduced conditions and shifted to repressing (R) or inducing (I) conditions before proceeding to protein extraction and Western blot analyses using anti-GFP antibodies or anti-actin as loading control. B) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH as in legend to Figure 6. C and D) Representative images of PalC-GFP localization of wild type and mutants under alkaline conditions. Bars, 5 μm.
Mentions: We introduced, in the alcAp::palC-GFPtransgene, mutations leading to PalC Arg442Ala, Pro439Phe or Tyr451Ala substitutions or to the Arg442Δ in-frame deletion. None of these mutant transgenes complemented a phenotypically palC4allele in plate tests, which demonstrates that Arg442Ala and Tyr451Ala substitutions result in a loss of function phenotype (Figure S2). Western blot analyses showed that, under inducing conditions, Arg442Ala and Arg442Δ mutant transgenes synthesized full-length PalC-GFP proteins with steady-state levels similar to the wild type (Figure 7A, compare anti-GFP lanes 4 and 6 with lane 2). In contrast, Tyr451Ala and Pro439Phe PalC-GFP levels were lower (Figure 7A, lanes 8 and 10). Detection of degradation bands in the Tyr451Ala mutant indicates that this particular sequence change leads to decreased protein stability in Aspergillus.

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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