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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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PalC-GFP localizes to the cytosol and to peripheral punctate structures in a pH-dependent manner.A) An alcAp::PalC-GFPtransgene was introduced in single copy at the argBlocus. Spores were germinated under inducing conditions at acidic pH and transferred for 30 minutes to repressing conditions at either acidic (B) or alkaline (C and D) pH before imaging GFP fluorescence. B) Subcellular localization of PalC-GFP upon transfer to acidic conditions. A few peripheral punctate structures are noticeable, a situation which was also seen in control germlings imaged before the transfer to repressing conditions (not shown). C) Subcellular localization of PalC-GFP upon transfer to alkaline conditions. Note the marked increase in the abundance of punctate structures. D) A short germling showing the cortical localization of PalC-GFP puncta. E) Quantification of punctate structures in germlings containing the alcAp-driven transgene shifted to acidic or alkaline conditions. F–H) Phenotypic characterization of palC-GFPgene-replaced strains and subcellular localization of the fusion protein expressed at physiological levels. F) Diagnostic growth tests showing that two independent clones in which the resident palCopen reading frame has been replaced by an open reading frame encoding a PalC-GFP fusion are indistinguishable from the wild type. Acidity-mimicking loss of function mutations in palCtypically result in hypersensitivity to sodium molybdate and alkaline pH and resistance to neomycin. MCA is Aspergilluscomplete medium. G) Strains were cultured at acidic pH and shifted to the indicated pH conditions for 30 minutes before GFP imaging. No punctate structures were seen in untagged control strains at either pH (not shown). H) Quantification of punctate structures in germlings shifted to acidic, neutral or alkaline conditions. Because significant pH signalling takes place under neutral conditions, PalC-GFP punctate structures are nearly as abundant upon transfer to neutral as to alkaline conditions. Bars, 5 μm.
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fig06: PalC-GFP localizes to the cytosol and to peripheral punctate structures in a pH-dependent manner.A) An alcAp::PalC-GFPtransgene was introduced in single copy at the argBlocus. Spores were germinated under inducing conditions at acidic pH and transferred for 30 minutes to repressing conditions at either acidic (B) or alkaline (C and D) pH before imaging GFP fluorescence. B) Subcellular localization of PalC-GFP upon transfer to acidic conditions. A few peripheral punctate structures are noticeable, a situation which was also seen in control germlings imaged before the transfer to repressing conditions (not shown). C) Subcellular localization of PalC-GFP upon transfer to alkaline conditions. Note the marked increase in the abundance of punctate structures. D) A short germling showing the cortical localization of PalC-GFP puncta. E) Quantification of punctate structures in germlings containing the alcAp-driven transgene shifted to acidic or alkaline conditions. F–H) Phenotypic characterization of palC-GFPgene-replaced strains and subcellular localization of the fusion protein expressed at physiological levels. F) Diagnostic growth tests showing that two independent clones in which the resident palCopen reading frame has been replaced by an open reading frame encoding a PalC-GFP fusion are indistinguishable from the wild type. Acidity-mimicking loss of function mutations in palCtypically result in hypersensitivity to sodium molybdate and alkaline pH and resistance to neomycin. MCA is Aspergilluscomplete medium. G) Strains were cultured at acidic pH and shifted to the indicated pH conditions for 30 minutes before GFP imaging. No punctate structures were seen in untagged control strains at either pH (not shown). H) Quantification of punctate structures in germlings shifted to acidic, neutral or alkaline conditions. Because significant pH signalling takes place under neutral conditions, PalC-GFP punctate structures are nearly as abundant upon transfer to neutral as to alkaline conditions. Bars, 5 μm.

Mentions: We constructed transgenes encoding PalC tagged with GFP at the N- or C-terminus, under the control of the strong, ethanol inducible and glucose repressible alcA(alcohol dehydrogenase) gene promoter and introduced them in single copy at the argBlocus (Figure 6A). N-terminal GFP attachment abolished PalC function, as determined by the inability of the fusion protein to complement the palC4loss of function mutation (data not shown). In contrast, PalC-GFP was phenotypically indistinguishable from untagged PalC in its ability to complement palC4under alcAp inducing conditions (Figure S2). A strain expressing this fusion protein was thus used in epifluorescence microscopy.


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)

PalC-GFP localizes to the cytosol and to peripheral punctate structures in a pH-dependent manner.A) An alcAp::PalC-GFPtransgene was introduced in single copy at the argBlocus. Spores were germinated under inducing conditions at acidic pH and transferred for 30 minutes to repressing conditions at either acidic (B) or alkaline (C and D) pH before imaging GFP fluorescence. B) Subcellular localization of PalC-GFP upon transfer to acidic conditions. A few peripheral punctate structures are noticeable, a situation which was also seen in control germlings imaged before the transfer to repressing conditions (not shown). C) Subcellular localization of PalC-GFP upon transfer to alkaline conditions. Note the marked increase in the abundance of punctate structures. D) A short germling showing the cortical localization of PalC-GFP puncta. E) Quantification of punctate structures in germlings containing the alcAp-driven transgene shifted to acidic or alkaline conditions. F–H) Phenotypic characterization of palC-GFPgene-replaced strains and subcellular localization of the fusion protein expressed at physiological levels. F) Diagnostic growth tests showing that two independent clones in which the resident palCopen reading frame has been replaced by an open reading frame encoding a PalC-GFP fusion are indistinguishable from the wild type. Acidity-mimicking loss of function mutations in palCtypically result in hypersensitivity to sodium molybdate and alkaline pH and resistance to neomycin. MCA is Aspergilluscomplete medium. G) Strains were cultured at acidic pH and shifted to the indicated pH conditions for 30 minutes before GFP imaging. No punctate structures were seen in untagged control strains at either pH (not shown). H) Quantification of punctate structures in germlings shifted to acidic, neutral or alkaline conditions. Because significant pH signalling takes place under neutral conditions, PalC-GFP punctate structures are nearly as abundant upon transfer to neutral as to alkaline conditions. Bars, 5 μm.
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Related In: Results  -  Collection

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fig06: PalC-GFP localizes to the cytosol and to peripheral punctate structures in a pH-dependent manner.A) An alcAp::PalC-GFPtransgene was introduced in single copy at the argBlocus. Spores were germinated under inducing conditions at acidic pH and transferred for 30 minutes to repressing conditions at either acidic (B) or alkaline (C and D) pH before imaging GFP fluorescence. B) Subcellular localization of PalC-GFP upon transfer to acidic conditions. A few peripheral punctate structures are noticeable, a situation which was also seen in control germlings imaged before the transfer to repressing conditions (not shown). C) Subcellular localization of PalC-GFP upon transfer to alkaline conditions. Note the marked increase in the abundance of punctate structures. D) A short germling showing the cortical localization of PalC-GFP puncta. E) Quantification of punctate structures in germlings containing the alcAp-driven transgene shifted to acidic or alkaline conditions. F–H) Phenotypic characterization of palC-GFPgene-replaced strains and subcellular localization of the fusion protein expressed at physiological levels. F) Diagnostic growth tests showing that two independent clones in which the resident palCopen reading frame has been replaced by an open reading frame encoding a PalC-GFP fusion are indistinguishable from the wild type. Acidity-mimicking loss of function mutations in palCtypically result in hypersensitivity to sodium molybdate and alkaline pH and resistance to neomycin. MCA is Aspergilluscomplete medium. G) Strains were cultured at acidic pH and shifted to the indicated pH conditions for 30 minutes before GFP imaging. No punctate structures were seen in untagged control strains at either pH (not shown). H) Quantification of punctate structures in germlings shifted to acidic, neutral or alkaline conditions. Because significant pH signalling takes place under neutral conditions, PalC-GFP punctate structures are nearly as abundant upon transfer to neutral as to alkaline conditions. Bars, 5 μm.
Mentions: We constructed transgenes encoding PalC tagged with GFP at the N- or C-terminus, under the control of the strong, ethanol inducible and glucose repressible alcA(alcohol dehydrogenase) gene promoter and introduced them in single copy at the argBlocus (Figure 6A). N-terminal GFP attachment abolished PalC function, as determined by the inability of the fusion protein to complement the palC4loss of function mutation (data not shown). In contrast, PalC-GFP was phenotypically indistinguishable from untagged PalC in its ability to complement palC4under alcAp inducing conditions (Figure S2). A strain expressing this fusion protein was thus used in epifluorescence microscopy.

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