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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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PalH-dependent and PalA-independent localization of PalC to cortical punctate structures.Strains carrying the gene-replaced palC-GFPallele in combination with palA34or palH72were compared with palC-GFPwild-type controls. A) Western blot analyses, using anti-GFP and anti-actin antibodies, of protein extracts from the wild type and from mutant strains (two clones with the indicated genotypes were analysed among the progeny of each cross). B and C) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH. D) Representative images of PalC-GFP localization in the wild type and in the indicated mutants under acidic and alkaline conditions. Bars, 5 μm.
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fig08: PalH-dependent and PalA-independent localization of PalC to cortical punctate structures.Strains carrying the gene-replaced palC-GFPallele in combination with palA34or palH72were compared with palC-GFPwild-type controls. A) Western blot analyses, using anti-GFP and anti-actin antibodies, of protein extracts from the wild type and from mutant strains (two clones with the indicated genotypes were analysed among the progeny of each cross). B and C) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH. D) Representative images of PalC-GFP localization in the wild type and in the indicated mutants under acidic and alkaline conditions. Bars, 5 μm.

Mentions: As PalC localization to cortical punctate structures appears to be independent of Vps32, but dependent on ambient pH, we tested whether this localization requires the 7-TMD receptor and likely pH sensor PalH. The palH72mutation truncates the 760 residue after residue 12 (S. Negrete-Urtasun, E. A. Espeso, H. N. A. and M. A. P., Imperial College London and Centro de Investigaciones Biológicas Madrid, unpublished results). We constructed by meiotic recombination a strain carrying palH72, in which PalC-GFP is the only functional PalC, for comparison with the corresponding palH+ strain. The genotype of this strain was confirmed by direct sequencing of the mutant palH72allele. Western blotting demonstrated that full-length PalC-GFP was synthesized irrespective of the presence or absence of the palH72 mutation (Figure 8A). Both palH+ and palH72strains showed predominant cytosolic fluorescence under acidic conditions. However, when cells were shifted to alkaline pH, palH72prevented the characteristic wild-type PalC-GFP localization to cortical puncta (Figure 8B,D).


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)

PalH-dependent and PalA-independent localization of PalC to cortical punctate structures.Strains carrying the gene-replaced palC-GFPallele in combination with palA34or palH72were compared with palC-GFPwild-type controls. A) Western blot analyses, using anti-GFP and anti-actin antibodies, of protein extracts from the wild type and from mutant strains (two clones with the indicated genotypes were analysed among the progeny of each cross). B and C) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH. D) Representative images of PalC-GFP localization in the wild type and in the indicated mutants under acidic and alkaline conditions. Bars, 5 μm.
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Related In: Results  -  Collection

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fig08: PalH-dependent and PalA-independent localization of PalC to cortical punctate structures.Strains carrying the gene-replaced palC-GFPallele in combination with palA34or palH72were compared with palC-GFPwild-type controls. A) Western blot analyses, using anti-GFP and anti-actin antibodies, of protein extracts from the wild type and from mutant strains (two clones with the indicated genotypes were analysed among the progeny of each cross). B and C) Quantification of PalC-GFP punctate structures upon shifting the indicated strains to alkaline pH. D) Representative images of PalC-GFP localization in the wild type and in the indicated mutants under acidic and alkaline conditions. Bars, 5 μm.
Mentions: As PalC localization to cortical punctate structures appears to be independent of Vps32, but dependent on ambient pH, we tested whether this localization requires the 7-TMD receptor and likely pH sensor PalH. The palH72mutation truncates the 760 residue after residue 12 (S. Negrete-Urtasun, E. A. Espeso, H. N. A. and M. A. P., Imperial College London and Centro de Investigaciones Biológicas Madrid, unpublished results). We constructed by meiotic recombination a strain carrying palH72, in which PalC-GFP is the only functional PalC, for comparison with the corresponding palH+ strain. The genotype of this strain was confirmed by direct sequencing of the mutant palH72allele. Western blotting demonstrated that full-length PalC-GFP was synthesized irrespective of the presence or absence of the palH72 mutation (Figure 8A). Both palH+ and palH72strains showed predominant cytosolic fluorescence under acidic conditions. However, when cells were shifted to alkaline pH, palH72prevented the characteristic wild-type PalC-GFP localization to cortical puncta (Figure 8B,D).

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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