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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 is absolutely required for pH signalling.Western blot analysis of Myc-tagged PacC two step proteolytic processing in palC+ and palC4 cells cultured under acidic conditions and shifted to alkaline conditions. The positions of PacC72, PacC53 and PacC27 are indicated. The loss of function mutation palC4prevents the signalling protease cleavage converting PacC72 to PacC53 (and thus prevents processing proteolysis to PacC27) (35). Note the previously reported markedly abnormal electrophoretic mobility of the PacC forms in SDS gels, possibly resulting from the highly basic N-terminal zinc-finger region (51).
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fig01: PalC is absolutely required for pH signalling.Western blot analysis of Myc-tagged PacC two step proteolytic processing in palC+ and palC4 cells cultured under acidic conditions and shifted to alkaline conditions. The positions of PacC72, PacC53 and PacC27 are indicated. The loss of function mutation palC4prevents the signalling protease cleavage converting PacC72 to PacC53 (and thus prevents processing proteolysis to PacC27) (35). Note the previously reported markedly abnormal electrophoretic mobility of the PacC forms in SDS gels, possibly resulting from the highly basic N-terminal zinc-finger region (51).

Mentions: When wild-type A. nidulanscells are transferred from acidic to alkaline pH, full-length PacC72 is activated through a two-step proteolytic processing resulting in the conversion of PacC72 to the intermediate PacC53 and the PacC27 final product. palC4, a phenotypically mutation, truncates the protein after residue 365 (33), within the Bro1 domain similarity region (see below). In mutant palC4cells, PacC72 remains unprocessed upon transfer to alkaline conditions (Figure 1), similarly to mutant palA−(35) and palB−(36) cells. Thus, PalC is absolutely required for the pH-dependent proteolytic activation of PacC.


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 is absolutely required for pH signalling.Western blot analysis of Myc-tagged PacC two step proteolytic processing in palC+ and palC4 cells cultured under acidic conditions and shifted to alkaline conditions. The positions of PacC72, PacC53 and PacC27 are indicated. The loss of function mutation palC4prevents the signalling protease cleavage converting PacC72 to PacC53 (and thus prevents processing proteolysis to PacC27) (35). Note the previously reported markedly abnormal electrophoretic mobility of the PacC forms in SDS gels, possibly resulting from the highly basic N-terminal zinc-finger region (51).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171039&req=5

fig01: PalC is absolutely required for pH signalling.Western blot analysis of Myc-tagged PacC two step proteolytic processing in palC+ and palC4 cells cultured under acidic conditions and shifted to alkaline conditions. The positions of PacC72, PacC53 and PacC27 are indicated. The loss of function mutation palC4prevents the signalling protease cleavage converting PacC72 to PacC53 (and thus prevents processing proteolysis to PacC27) (35). Note the previously reported markedly abnormal electrophoretic mobility of the PacC forms in SDS gels, possibly resulting from the highly basic N-terminal zinc-finger region (51).
Mentions: When wild-type A. nidulanscells are transferred from acidic to alkaline pH, full-length PacC72 is activated through a two-step proteolytic processing resulting in the conversion of PacC72 to the intermediate PacC53 and the PacC27 final product. palC4, a phenotypically mutation, truncates the protein after residue 365 (33), within the Bro1 domain similarity region (see below). In mutant palC4cells, PacC72 remains unprocessed upon transfer to alkaline conditions (Figure 1), similarly to mutant palA−(35) and palB−(36) cells. Thus, PalC is absolutely required for the pH-dependent proteolytic activation of PacC.

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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