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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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Characterization of PalC-GFP and Vps32-GFP-containing compartments.A) PalC-GFP punctate structures and FM4-64-stained plasma membrane and associated cortical structures stained shortly after dye loading. Examples of cortical and nearly cortical PalC-GFP punctate structures that do not associate with cortical FM4-64 puncta are indicated by empty and filled arrowheads, respectively. Thick arrows indicate two examples of FM4-64 cortical structures that do not associate with PalC-GFP. Thin arrows indicate two (rare) examples where PalC-GFP and FM4-64 cortical structures are closely associated. B and C) PalC-GFP punctate structures do not associate with endomembranes stained with FM4-64. B) One of several vacuoles labelled by FM4-64 after a 45-minute chase (see text) in the swelled basal conidiospore is arrowed. C) Endomembranes stained with FM4-64 after a 45-minute chase include a network of mitochondria and endoplasmic reticulum (ER). One nucleus showing labelling of its ER-associated membrane is arrowed. D) PalC-GFP punctate structures do not associate with AbpA-mRFP-labelled actin patches. E) Motile FM4-64 early endosomes filmed within 5 minutes after dye loading in an incubation chamber at 28°C. Frames were taken from Movie S2, which was made using the stream acquisition feature of the MetaMorph Universal Imaging software with 0.1 seconds exposures. Elapsed time is indicated in seconds:milliseconds. One of the several endosomes moving very near the cortex is indicated with an arrowhead. A second endosome moving in the opposite direction and stopping in the vicinity of a nucleus is arrowed. A larger, static endosome is indicated with an asterisk. Bar, 5 μm. F) GST-PalC pulls down Vps32-GFP but not GFP-Rab5 or GFP from Aspergillus nidulansprotein extracts from cells expressing the indicated proteins. Glutathione S-transferase does not pull down Vps32-GFP. Glutathione–Sepharose bound proteins were run in twin 10% polyacrylamide gels, one of which was analysed by Western blot using an anti-GFP antibody (α-GFP), whereas the second was stained with Coomassie Blue to show the amounts of GST-PalC and GST baits used in the different lanes. G) Motile endosomes labelled with Vps32-GFP. Frames, shown in inverted contrast, were taken from Movie S3, which should be consulted to get a more accurate record of endosome motility and trajectories. The movie was made using the stream acquisition feature of MetaMorph with 0.5 seconds exposures and a 2 × 2 binning. Elapsed time is indicated in seconds:milliseconds. Endosomes showing retrograde and anterograde movement (two of each class) are indicated with arrowed numbers. Note that subapical ‘black’ endosome 2 appears to be formed after receiving traffic from ‘white’ endosomes 1 and 2 moving in anterograde direction. The dotted arrow indicates a static endosome, which may be used as reference.
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fig09: Characterization of PalC-GFP and Vps32-GFP-containing compartments.A) PalC-GFP punctate structures and FM4-64-stained plasma membrane and associated cortical structures stained shortly after dye loading. Examples of cortical and nearly cortical PalC-GFP punctate structures that do not associate with cortical FM4-64 puncta are indicated by empty and filled arrowheads, respectively. Thick arrows indicate two examples of FM4-64 cortical structures that do not associate with PalC-GFP. Thin arrows indicate two (rare) examples where PalC-GFP and FM4-64 cortical structures are closely associated. B and C) PalC-GFP punctate structures do not associate with endomembranes stained with FM4-64. B) One of several vacuoles labelled by FM4-64 after a 45-minute chase (see text) in the swelled basal conidiospore is arrowed. C) Endomembranes stained with FM4-64 after a 45-minute chase include a network of mitochondria and endoplasmic reticulum (ER). One nucleus showing labelling of its ER-associated membrane is arrowed. D) PalC-GFP punctate structures do not associate with AbpA-mRFP-labelled actin patches. E) Motile FM4-64 early endosomes filmed within 5 minutes after dye loading in an incubation chamber at 28°C. Frames were taken from Movie S2, which was made using the stream acquisition feature of the MetaMorph Universal Imaging software with 0.1 seconds exposures. Elapsed time is indicated in seconds:milliseconds. One of the several endosomes moving very near the cortex is indicated with an arrowhead. A second endosome moving in the opposite direction and stopping in the vicinity of a nucleus is arrowed. A larger, static endosome is indicated with an asterisk. Bar, 5 μm. F) GST-PalC pulls down Vps32-GFP but not GFP-Rab5 or GFP from Aspergillus nidulansprotein extracts from cells expressing the indicated proteins. Glutathione S-transferase does not pull down Vps32-GFP. Glutathione–Sepharose bound proteins were run in twin 10% polyacrylamide gels, one of which was analysed by Western blot using an anti-GFP antibody (α-GFP), whereas the second was stained with Coomassie Blue to show the amounts of GST-PalC and GST baits used in the different lanes. G) Motile endosomes labelled with Vps32-GFP. Frames, shown in inverted contrast, were taken from Movie S3, which should be consulted to get a more accurate record of endosome motility and trajectories. The movie was made using the stream acquisition feature of MetaMorph with 0.5 seconds exposures and a 2 × 2 binning. Elapsed time is indicated in seconds:milliseconds. Endosomes showing retrograde and anterograde movement (two of each class) are indicated with arrowed numbers. Note that subapical ‘black’ endosome 2 appears to be formed after receiving traffic from ‘white’ endosomes 1 and 2 moving in anterograde direction. The dotted arrow indicates a static endosome, which may be used as reference.

Mentions: PalC-GFP is recruited to cortical punctate structures very rapidly after a pH shift, within the time required to mount germlings on microscopy slides and start fluorescent image acquisition. Using time-lapse fluorescence microscopy, we determined that these structures are static (Movie S1). We attempted a preliminary characterization of these cortical sites where PalC is recruited using the lipophilic fluorescent dye FM4-64. Within a 5-minute chase after a cold loading of FM4-64 (42), the dye labels the plasma membrane and cortical punctate structures that possibly represent sites of lipid internalization. Double-label experiments demonstrated that PalC-GFP cortical structures colocalize or closely associate with the plasma membrane (Figure 9A, empty and filled arrowheads, respectively). FM4-64 and PalC-GFP cortical punctate structures occasionally overlap (Figure 9A, thin arrows), although in a majority of cases they do not associate (e.g. Figure 9A, thick arrows). After a 45-minute chase, FM4-64 is found in vacuolar membranes (Figure 9B, arrow) and in a network of endomembranes containing mitochondria and endoplasmic reticulum (Figure 9B,C) (42). PalC-GFP-containing structures do not associate with either (Figure 9B,C; the position of a nucleus in panel C is indicated with an arrow). As in S. cerevisiae, the A. nidulansactin cytoskeleton involves actin cables and cortical patches (43). To determine whether PalC-GFP structures associate with cortical actin patches, we made double-labelling experiments with AbpA-monomeric red fluorescence protein (mRFP). AbpA (the orthologue of yeast Abp1p) is a prototypic marker of these sites (L. Araujo-Bazán, M. A. P. and E. Espeso, submitted). PalC-GFP and AbpA-mRFP do not colocalize. AbpA patches are slightly subcortical and most of them are seen in a different focal plane than PalC-GFP (Figure 9D).


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)

Characterization of PalC-GFP and Vps32-GFP-containing compartments.A) PalC-GFP punctate structures and FM4-64-stained plasma membrane and associated cortical structures stained shortly after dye loading. Examples of cortical and nearly cortical PalC-GFP punctate structures that do not associate with cortical FM4-64 puncta are indicated by empty and filled arrowheads, respectively. Thick arrows indicate two examples of FM4-64 cortical structures that do not associate with PalC-GFP. Thin arrows indicate two (rare) examples where PalC-GFP and FM4-64 cortical structures are closely associated. B and C) PalC-GFP punctate structures do not associate with endomembranes stained with FM4-64. B) One of several vacuoles labelled by FM4-64 after a 45-minute chase (see text) in the swelled basal conidiospore is arrowed. C) Endomembranes stained with FM4-64 after a 45-minute chase include a network of mitochondria and endoplasmic reticulum (ER). One nucleus showing labelling of its ER-associated membrane is arrowed. D) PalC-GFP punctate structures do not associate with AbpA-mRFP-labelled actin patches. E) Motile FM4-64 early endosomes filmed within 5 minutes after dye loading in an incubation chamber at 28°C. Frames were taken from Movie S2, which was made using the stream acquisition feature of the MetaMorph Universal Imaging software with 0.1 seconds exposures. Elapsed time is indicated in seconds:milliseconds. One of the several endosomes moving very near the cortex is indicated with an arrowhead. A second endosome moving in the opposite direction and stopping in the vicinity of a nucleus is arrowed. A larger, static endosome is indicated with an asterisk. Bar, 5 μm. F) GST-PalC pulls down Vps32-GFP but not GFP-Rab5 or GFP from Aspergillus nidulansprotein extracts from cells expressing the indicated proteins. Glutathione S-transferase does not pull down Vps32-GFP. Glutathione–Sepharose bound proteins were run in twin 10% polyacrylamide gels, one of which was analysed by Western blot using an anti-GFP antibody (α-GFP), whereas the second was stained with Coomassie Blue to show the amounts of GST-PalC and GST baits used in the different lanes. G) Motile endosomes labelled with Vps32-GFP. Frames, shown in inverted contrast, were taken from Movie S3, which should be consulted to get a more accurate record of endosome motility and trajectories. The movie was made using the stream acquisition feature of MetaMorph with 0.5 seconds exposures and a 2 × 2 binning. Elapsed time is indicated in seconds:milliseconds. Endosomes showing retrograde and anterograde movement (two of each class) are indicated with arrowed numbers. Note that subapical ‘black’ endosome 2 appears to be formed after receiving traffic from ‘white’ endosomes 1 and 2 moving in anterograde direction. The dotted arrow indicates a static endosome, which may be used as reference.
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fig09: Characterization of PalC-GFP and Vps32-GFP-containing compartments.A) PalC-GFP punctate structures and FM4-64-stained plasma membrane and associated cortical structures stained shortly after dye loading. Examples of cortical and nearly cortical PalC-GFP punctate structures that do not associate with cortical FM4-64 puncta are indicated by empty and filled arrowheads, respectively. Thick arrows indicate two examples of FM4-64 cortical structures that do not associate with PalC-GFP. Thin arrows indicate two (rare) examples where PalC-GFP and FM4-64 cortical structures are closely associated. B and C) PalC-GFP punctate structures do not associate with endomembranes stained with FM4-64. B) One of several vacuoles labelled by FM4-64 after a 45-minute chase (see text) in the swelled basal conidiospore is arrowed. C) Endomembranes stained with FM4-64 after a 45-minute chase include a network of mitochondria and endoplasmic reticulum (ER). One nucleus showing labelling of its ER-associated membrane is arrowed. D) PalC-GFP punctate structures do not associate with AbpA-mRFP-labelled actin patches. E) Motile FM4-64 early endosomes filmed within 5 minutes after dye loading in an incubation chamber at 28°C. Frames were taken from Movie S2, which was made using the stream acquisition feature of the MetaMorph Universal Imaging software with 0.1 seconds exposures. Elapsed time is indicated in seconds:milliseconds. One of the several endosomes moving very near the cortex is indicated with an arrowhead. A second endosome moving in the opposite direction and stopping in the vicinity of a nucleus is arrowed. A larger, static endosome is indicated with an asterisk. Bar, 5 μm. F) GST-PalC pulls down Vps32-GFP but not GFP-Rab5 or GFP from Aspergillus nidulansprotein extracts from cells expressing the indicated proteins. Glutathione S-transferase does not pull down Vps32-GFP. Glutathione–Sepharose bound proteins were run in twin 10% polyacrylamide gels, one of which was analysed by Western blot using an anti-GFP antibody (α-GFP), whereas the second was stained with Coomassie Blue to show the amounts of GST-PalC and GST baits used in the different lanes. G) Motile endosomes labelled with Vps32-GFP. Frames, shown in inverted contrast, were taken from Movie S3, which should be consulted to get a more accurate record of endosome motility and trajectories. The movie was made using the stream acquisition feature of MetaMorph with 0.5 seconds exposures and a 2 × 2 binning. Elapsed time is indicated in seconds:milliseconds. Endosomes showing retrograde and anterograde movement (two of each class) are indicated with arrowed numbers. Note that subapical ‘black’ endosome 2 appears to be formed after receiving traffic from ‘white’ endosomes 1 and 2 moving in anterograde direction. The dotted arrow indicates a static endosome, which may be used as reference.
Mentions: PalC-GFP is recruited to cortical punctate structures very rapidly after a pH shift, within the time required to mount germlings on microscopy slides and start fluorescent image acquisition. Using time-lapse fluorescence microscopy, we determined that these structures are static (Movie S1). We attempted a preliminary characterization of these cortical sites where PalC is recruited using the lipophilic fluorescent dye FM4-64. Within a 5-minute chase after a cold loading of FM4-64 (42), the dye labels the plasma membrane and cortical punctate structures that possibly represent sites of lipid internalization. Double-label experiments demonstrated that PalC-GFP cortical structures colocalize or closely associate with the plasma membrane (Figure 9A, empty and filled arrowheads, respectively). FM4-64 and PalC-GFP cortical punctate structures occasionally overlap (Figure 9A, thin arrows), although in a majority of cases they do not associate (e.g. Figure 9A, thick arrows). After a 45-minute chase, FM4-64 is found in vacuolar membranes (Figure 9B, arrow) and in a network of endomembranes containing mitochondria and endoplasmic reticulum (Figure 9B,C) (42). PalC-GFP-containing structures do not associate with either (Figure 9B,C; the position of a nucleus in panel C is indicated with an arrow). As in S. cerevisiae, the A. nidulansactin cytoskeleton involves actin cables and cortical patches (43). To determine whether PalC-GFP structures associate with cortical actin patches, we made double-labelling experiments with AbpA-monomeric red fluorescence protein (mRFP). AbpA (the orthologue of yeast Abp1p) is a prototypic marker of these sites (L. Araujo-Bazán, M. A. P. and E. Espeso, submitted). PalC-GFP and AbpA-mRFP do not colocalize. AbpA patches are slightly subcortical and most of them are seen in a different focal plane than PalC-GFP (Figure 9D).

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