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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 interacts directly with Vps32 and this interaction involves Arg442 within conserved region I.A) GST-PalC (white diamond) and zz-Vps32 (white star) fusion proteins expressed in E. colicopurify on glutathione–Sepharose beads (GST pull downs) after mixing the corresponding bacterial extracts (lanes labelled ‘in’, inputs). KapA50 (white square) is a 50-kDa truncated version of the A. nidulansKapA importin α lacking the auto-inhibitory domain. NapBVps75 (white circle) is the A. nidulanshomologue of Saccharomyces cerevisiaeVps75p. The closed arrow indicates the mobility of zz-Vps32 copurifying with GST-PalC. B) GST–PalC interaction with zz-Vps32 resists NaCl up to 300 mm but is disrupted by 500 mm NaCl. C) zz-Vps25 does not copurify with GST-PalC, whose interaction with zz-Vps32 is prevented by Arg442Ala, Arg442His and Arg442Δ. In A and C, the NaCl concentration was 200 mm.
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fig05: PalC interacts directly with Vps32 and this interaction involves Arg442 within conserved region I.A) GST-PalC (white diamond) and zz-Vps32 (white star) fusion proteins expressed in E. colicopurify on glutathione–Sepharose beads (GST pull downs) after mixing the corresponding bacterial extracts (lanes labelled ‘in’, inputs). KapA50 (white square) is a 50-kDa truncated version of the A. nidulansKapA importin α lacking the auto-inhibitory domain. NapBVps75 (white circle) is the A. nidulanshomologue of Saccharomyces cerevisiaeVps75p. The closed arrow indicates the mobility of zz-Vps32 copurifying with GST-PalC. B) GST–PalC interaction with zz-Vps32 resists NaCl up to 300 mm but is disrupted by 500 mm NaCl. C) zz-Vps25 does not copurify with GST-PalC, whose interaction with zz-Vps32 is prevented by Arg442Ala, Arg442His and Arg442Δ. In A and C, the NaCl concentration was 200 mm.

Mentions: To determine whether the PalC–Vps32 interaction is direct and to rule out the possibility that the presence of N-terminal Gal4p DNA binding and activation domains in Vps32 two-hybrid assay fusion proteins alters the interactive properties of Vps32, we bacterially expressed a glutathione S-transferase (GST)::PalC fusion protein for pull-down experiments with glutathione–Sepharose beads (Figure 5A). GST::PalC efficiently and specifically pulls down zz-Vps32 from a bacterial extract (Figure 5, lane 5). (zz indicates a tandem repetition of a ‘z’ synthetic immunoglobulin-G (IgG)-binding domain of protein A). This interaction is resistant to relatively high ionic strength, as it is prevented only by 0.5 m NaCl but not by 0.3 m NaCl or salt concentrations below that (Figure 5B). In contrast, GST::PalC did not pull down the unrelated protein KapA50 (Figure 5A, lane 6) nor did the unrelated GST-NapB fusion protein control pull-down Vps32. Because GST::PalC does not pull down the ESCRT-II component Vps25 either (Figure 5C, lines 1 and 2), we conclude that PalC interacts specifically and directly with Vps32. Under conditions in which glutathione–Sepharose beads were not saturated with GST::PalC (data not shown), quantitative analysis of digitized images indicated that 0.9 mol of zz-Vps32 was pulled down per mole of GST::PalC.


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 interacts directly with Vps32 and this interaction involves Arg442 within conserved region I.A) GST-PalC (white diamond) and zz-Vps32 (white star) fusion proteins expressed in E. colicopurify on glutathione–Sepharose beads (GST pull downs) after mixing the corresponding bacterial extracts (lanes labelled ‘in’, inputs). KapA50 (white square) is a 50-kDa truncated version of the A. nidulansKapA importin α lacking the auto-inhibitory domain. NapBVps75 (white circle) is the A. nidulanshomologue of Saccharomyces cerevisiaeVps75p. The closed arrow indicates the mobility of zz-Vps32 copurifying with GST-PalC. B) GST–PalC interaction with zz-Vps32 resists NaCl up to 300 mm but is disrupted by 500 mm NaCl. C) zz-Vps25 does not copurify with GST-PalC, whose interaction with zz-Vps32 is prevented by Arg442Ala, Arg442His and Arg442Δ. In A and C, the NaCl concentration was 200 mm.
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fig05: PalC interacts directly with Vps32 and this interaction involves Arg442 within conserved region I.A) GST-PalC (white diamond) and zz-Vps32 (white star) fusion proteins expressed in E. colicopurify on glutathione–Sepharose beads (GST pull downs) after mixing the corresponding bacterial extracts (lanes labelled ‘in’, inputs). KapA50 (white square) is a 50-kDa truncated version of the A. nidulansKapA importin α lacking the auto-inhibitory domain. NapBVps75 (white circle) is the A. nidulanshomologue of Saccharomyces cerevisiaeVps75p. The closed arrow indicates the mobility of zz-Vps32 copurifying with GST-PalC. B) GST–PalC interaction with zz-Vps32 resists NaCl up to 300 mm but is disrupted by 500 mm NaCl. C) zz-Vps25 does not copurify with GST-PalC, whose interaction with zz-Vps32 is prevented by Arg442Ala, Arg442His and Arg442Δ. In A and C, the NaCl concentration was 200 mm.
Mentions: To determine whether the PalC–Vps32 interaction is direct and to rule out the possibility that the presence of N-terminal Gal4p DNA binding and activation domains in Vps32 two-hybrid assay fusion proteins alters the interactive properties of Vps32, we bacterially expressed a glutathione S-transferase (GST)::PalC fusion protein for pull-down experiments with glutathione–Sepharose beads (Figure 5A). GST::PalC efficiently and specifically pulls down zz-Vps32 from a bacterial extract (Figure 5, lane 5). (zz indicates a tandem repetition of a ‘z’ synthetic immunoglobulin-G (IgG)-binding domain of protein A). This interaction is resistant to relatively high ionic strength, as it is prevented only by 0.5 m NaCl but not by 0.3 m NaCl or salt concentrations below that (Figure 5B). In contrast, GST::PalC did not pull down the unrelated protein KapA50 (Figure 5A, lane 6) nor did the unrelated GST-NapB fusion protein control pull-down Vps32. Because GST::PalC does not pull down the ESCRT-II component Vps25 either (Figure 5C, lines 1 and 2), we conclude that PalC interacts specifically and directly with Vps32. Under conditions in which glutathione–Sepharose beads were not saturated with GST::PalC (data not shown), quantitative analysis of digitized images indicated that 0.9 mol of zz-Vps32 was pulled down per mole of GST::PalC.

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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