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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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The Bro1 domain in PalC. Triple amino acid sequence alignment of Saccharomyces cerevisiaeBro1p with its Aspergillus nidulansorthologue BroA and with PalC.Shown are Bro1p residues 1 through 400, beyond which no similarity with PalC is discernable. Bro1p residues 1–387 are included in the three-dimensional structure reported by Kim et al. (37). B and H indicate Bro1p residues in β-strands (green) and α-helices (blue), respectively. Note that the positions of secondary structure elements, taken from the authors’ secondary structure assignment shown in the Protein Data Bank entry 1ZB1, are slightly different from those in (37). Underlined PalC residues indicate regions located between predicted α-helical structure. The two conserved regions discussed in the text are bracketed. The position of PalC single-residue substitutions/deletion used in this work is indicated with blue triangles. Also indicated are the positions of PalC truncations corresponding to palC131(S455fs, blue dot) and palC179(L427stop, red dot) resulting in partial and complete loss of function, respectively (34). Residues conserved in all three proteins (according to the Blosum62 matrix) are highlighted in red, whereas those conserved in only two entries are highlighted in grey. Dashes indicate gaps. Residue numbers are indicated for Bro1p and PalC, above or below the respective sequences.
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fig02: The Bro1 domain in PalC. Triple amino acid sequence alignment of Saccharomyces cerevisiaeBro1p with its Aspergillus nidulansorthologue BroA and with PalC.Shown are Bro1p residues 1 through 400, beyond which no similarity with PalC is discernable. Bro1p residues 1–387 are included in the three-dimensional structure reported by Kim et al. (37). B and H indicate Bro1p residues in β-strands (green) and α-helices (blue), respectively. Note that the positions of secondary structure elements, taken from the authors’ secondary structure assignment shown in the Protein Data Bank entry 1ZB1, are slightly different from those in (37). Underlined PalC residues indicate regions located between predicted α-helical structure. The two conserved regions discussed in the text are bracketed. The position of PalC single-residue substitutions/deletion used in this work is indicated with blue triangles. Also indicated are the positions of PalC truncations corresponding to palC131(S455fs, blue dot) and palC179(L427stop, red dot) resulting in partial and complete loss of function, respectively (34). Residues conserved in all three proteins (according to the Blosum62 matrix) are highlighted in red, whereas those conserved in only two entries are highlighted in grey. Dashes indicate gaps. Residue numbers are indicated for Bro1p and PalC, above or below the respective sequences.

Mentions: Figure 2 shows a sequence alignment of a region encompassing the Bro1 domains of the prototype S. cerevisiaeBro1p/Vps31p, its A. nidulansorthologue BroA and PalC, with indication of secondary structure elements in Bro1p. Sequence similarity is rather conspicuous in several regions within the crystallographically determined Bro1 domain in Bro1p (37) and actually extends ∼30 residues beyond it, strongly supporting our previous observation (34) that PalC contains one such domain, albeit rather distant from those in BroA/Bro1p and its PalA/Rim20p paralogues. The Bro1 domain structure is mostly made of α-helices forming a single 11-helical solenoid (37), with helices 6 through 11 arranged in a tetratricopeptide-repeat-like substructure involving three paired helices and a C-terminal unpaired one (α-helix 11). While a significant proportion of sequence divergence between PalC and Bro1p roughly involves insertions between or at the borders of α-helical regions, the highest similarity is apparent in regions corresponding to Bro1p α-helices. Notable exceptions are two highly conserved regions downstream of Bro1p residue 312 (Figure 2). Conserved region I, partially overlapping with α-helix 11, involves Bro1p residues 313–340, thus including Leu336, Ala338 and Ile339, located at the hydrophobic patch 1 in the Bro1p structure (37). This hydrophobic patch 1 is rimmed by polar, solvent exposed Bro1p residues including Lys340 (Figure 2). Conserved region II (Bro1p residues 367-394) is located downstream of the most C-terminal ordered residue and thus, structural information for it is not available. PalC contains, within this region, a Pro-rich motif with an internal Tyr that is required for pH signalling in vivo(34).


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)

The Bro1 domain in PalC. Triple amino acid sequence alignment of Saccharomyces cerevisiaeBro1p with its Aspergillus nidulansorthologue BroA and with PalC.Shown are Bro1p residues 1 through 400, beyond which no similarity with PalC is discernable. Bro1p residues 1–387 are included in the three-dimensional structure reported by Kim et al. (37). B and H indicate Bro1p residues in β-strands (green) and α-helices (blue), respectively. Note that the positions of secondary structure elements, taken from the authors’ secondary structure assignment shown in the Protein Data Bank entry 1ZB1, are slightly different from those in (37). Underlined PalC residues indicate regions located between predicted α-helical structure. The two conserved regions discussed in the text are bracketed. The position of PalC single-residue substitutions/deletion used in this work is indicated with blue triangles. Also indicated are the positions of PalC truncations corresponding to palC131(S455fs, blue dot) and palC179(L427stop, red dot) resulting in partial and complete loss of function, respectively (34). Residues conserved in all three proteins (according to the Blosum62 matrix) are highlighted in red, whereas those conserved in only two entries are highlighted in grey. Dashes indicate gaps. Residue numbers are indicated for Bro1p and PalC, above or below the respective sequences.
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Related In: Results  -  Collection

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fig02: The Bro1 domain in PalC. Triple amino acid sequence alignment of Saccharomyces cerevisiaeBro1p with its Aspergillus nidulansorthologue BroA and with PalC.Shown are Bro1p residues 1 through 400, beyond which no similarity with PalC is discernable. Bro1p residues 1–387 are included in the three-dimensional structure reported by Kim et al. (37). B and H indicate Bro1p residues in β-strands (green) and α-helices (blue), respectively. Note that the positions of secondary structure elements, taken from the authors’ secondary structure assignment shown in the Protein Data Bank entry 1ZB1, are slightly different from those in (37). Underlined PalC residues indicate regions located between predicted α-helical structure. The two conserved regions discussed in the text are bracketed. The position of PalC single-residue substitutions/deletion used in this work is indicated with blue triangles. Also indicated are the positions of PalC truncations corresponding to palC131(S455fs, blue dot) and palC179(L427stop, red dot) resulting in partial and complete loss of function, respectively (34). Residues conserved in all three proteins (according to the Blosum62 matrix) are highlighted in red, whereas those conserved in only two entries are highlighted in grey. Dashes indicate gaps. Residue numbers are indicated for Bro1p and PalC, above or below the respective sequences.
Mentions: Figure 2 shows a sequence alignment of a region encompassing the Bro1 domains of the prototype S. cerevisiaeBro1p/Vps31p, its A. nidulansorthologue BroA and PalC, with indication of secondary structure elements in Bro1p. Sequence similarity is rather conspicuous in several regions within the crystallographically determined Bro1 domain in Bro1p (37) and actually extends ∼30 residues beyond it, strongly supporting our previous observation (34) that PalC contains one such domain, albeit rather distant from those in BroA/Bro1p and its PalA/Rim20p paralogues. The Bro1 domain structure is mostly made of α-helices forming a single 11-helical solenoid (37), with helices 6 through 11 arranged in a tetratricopeptide-repeat-like substructure involving three paired helices and a C-terminal unpaired one (α-helix 11). While a significant proportion of sequence divergence between PalC and Bro1p roughly involves insertions between or at the borders of α-helical regions, the highest similarity is apparent in regions corresponding to Bro1p α-helices. Notable exceptions are two highly conserved regions downstream of Bro1p residue 312 (Figure 2). Conserved region I, partially overlapping with α-helix 11, involves Bro1p residues 313–340, thus including Leu336, Ala338 and Ile339, located at the hydrophobic patch 1 in the Bro1p structure (37). This hydrophobic patch 1 is rimmed by polar, solvent exposed Bro1p residues including Lys340 (Figure 2). Conserved region II (Bro1p residues 367-394) is located downstream of the most C-terminal ordered residue and thus, structural information for it is not available. PalC contains, within this region, a Pro-rich motif with an internal Tyr that is required for pH signalling in vivo(34).

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