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Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation.

Stawiecka-Mirota M, Pokrzywa W, Morvan J, Zoladek T, Haguenauer-Tsapis R, Urban-Grimal D, Morsomme P - Traffic (2007)

Bottom Line: This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen.Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting.Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS, Universités Paris VI et VII, Département de Biologie Cellulaire, 2 Place Jussieu 75005 Paris, France.

ABSTRACT
Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.

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Sna3p undergoes Ub-dependent MVB sorting.sna3Δand VPH1-GFP cells expressing Sna3p-6HA or Sna3p-4KR-6HA were grown to mid-exponential phase. A) Sna3p-6HA or Sna3p-4KR-6HA produced in sna3Δ cells were detected by immunoblotting of cell lysates with anti-HA antibodies (left panel) and immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies (right panel). B) The production of Sna3p-6HA or Sna3p-4KR-6HA in VPH1-GFP cells was stopped by adding cycloheximide (100 μg/mL). Sna3p-6HA or Sna3p-4KR-6HA was detected by immunoblotting of cell lysates with anti-HA antibodies at the times indicated after the addition of cycloheximide, while Vph1-GFP was detected with anti-GFP antibodies. C) Phenylmethylsulfonyl fluoride was added for 1 h toVPH1-GFP cells producing Sna3p-6HA or Sna3p-4KR-6HA before the cells were fixed and processed for immunofluorescence with monoclonal anti-HA and polyclonal anti-GFP antibodies followed by Texas Red-labeled donkey anti-mouse IgG and FITC-labeled donkey anti-rabbit IgG as described in Materials and Methods. Cells were examined for fluorescence (the HA signal was detected using the rhodamine filter set and the GFP signal using the FITC filter set) and vacuole by Nomarski optics. IP: immunoprecipitation, WB: western-blot.
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fig05: Sna3p undergoes Ub-dependent MVB sorting.sna3Δand VPH1-GFP cells expressing Sna3p-6HA or Sna3p-4KR-6HA were grown to mid-exponential phase. A) Sna3p-6HA or Sna3p-4KR-6HA produced in sna3Δ cells were detected by immunoblotting of cell lysates with anti-HA antibodies (left panel) and immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies (right panel). B) The production of Sna3p-6HA or Sna3p-4KR-6HA in VPH1-GFP cells was stopped by adding cycloheximide (100 μg/mL). Sna3p-6HA or Sna3p-4KR-6HA was detected by immunoblotting of cell lysates with anti-HA antibodies at the times indicated after the addition of cycloheximide, while Vph1-GFP was detected with anti-GFP antibodies. C) Phenylmethylsulfonyl fluoride was added for 1 h toVPH1-GFP cells producing Sna3p-6HA or Sna3p-4KR-6HA before the cells were fixed and processed for immunofluorescence with monoclonal anti-HA and polyclonal anti-GFP antibodies followed by Texas Red-labeled donkey anti-mouse IgG and FITC-labeled donkey anti-rabbit IgG as described in Materials and Methods. Cells were examined for fluorescence (the HA signal was detected using the rhodamine filter set and the GFP signal using the FITC filter set) and vacuole by Nomarski optics. IP: immunoprecipitation, WB: western-blot.

Mentions: We investigated the role of Sna3p ubiquitylation in its MVB sorting both by biochemical and by immunofluorescence approaches using Sna3p tagged with HA, which does not carry any Lys residue. As immunofluorescent data using Sna3p-HA gave a signal that was too faint, we used a chimeric Sna3p carrying 6HA to improve the signal. We first checked the ubiquitylation fate of the fusion protein. Immunoblot of Sna3Δ cells expressing Sna3p-6HA revealed above the main Sna3p-6HA signal a ladder of bands of higher molecular weight, corresponding to ubiquitylated bands as evidenced by immunoprecipitation with antibodies against HA, followed by immunoblot with anti-Ub antibodies (Figure 5A). Sna3p-6HA ubiquitylation was completely abolished after mutation of four Sna3p lysines on the fusion protein, as revealed by both direct immunoblots and immunoprecipitation using anti-HA antibodies, followed by immunoblot with anti-Ub antibodies (Figure 5A). Hence, Sna3p-6HA mutated or not on its Lys residues was an appropriate tool for our purpose. We analyzed the stability and localization of Sna3-6HA and its four Lys-to-Arg counterpart in cells expressing a GFP-tagged version of Vph1p, a membrane subunit of the vacuolar adenosine triphosphatase (ATPase). We first investigated the turnover of Sna3-6HA and Sna3p-4KR-6HA after inhibiting protein synthesis by adding cycloheximide to exponentially growing cells (Figure 5B). Addition of cycloheximide for 1 h caused apparently no loss of Sna3p-4KR-6HA, while we observed a significant decrease in the amount of Sna3p-6HA compared with the control protein Vph1-GFP. This result indicates that a version of Sna3p that cannot be ubiquitylated is protected against vacuolar degradation. Next, we determined the respective localization of the two proteins by indirect immunofluorescence microscopy. To facilitate localization studies, phenylmethylsulfonyl fluoride (PMSF) was added for 1 h in exponentially grown cells to inhibit vacuolar proteases before cells were fixed and processed for immunofluorescence with anti-HA and anti-GFP antibodies (Figure 5C). Sna3p-6HA was clearly evidenced in vacuole interior, as judged by colocalization of the HA-specific fluorescent signal with vacuoles, detected by Nomarski optics. In contrast, we observed that the HA-specific signal of Sna3p-4KR-6HA co-localized with the GFP-specific signal of the vacuolar membrane marker, Vph1-GFP. Both biochemical and immunofluorescence microscopy data thus strongly suggest that the Lys-to-Arg version of Sna3p is not targeted to the vacuole interior. Therefore, as described for other MVB cargoes, Sna3p ubiquitylation is clearly required for its MVB sorting.


Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation.

Stawiecka-Mirota M, Pokrzywa W, Morvan J, Zoladek T, Haguenauer-Tsapis R, Urban-Grimal D, Morsomme P - Traffic (2007)

Sna3p undergoes Ub-dependent MVB sorting.sna3Δand VPH1-GFP cells expressing Sna3p-6HA or Sna3p-4KR-6HA were grown to mid-exponential phase. A) Sna3p-6HA or Sna3p-4KR-6HA produced in sna3Δ cells were detected by immunoblotting of cell lysates with anti-HA antibodies (left panel) and immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies (right panel). B) The production of Sna3p-6HA or Sna3p-4KR-6HA in VPH1-GFP cells was stopped by adding cycloheximide (100 μg/mL). Sna3p-6HA or Sna3p-4KR-6HA was detected by immunoblotting of cell lysates with anti-HA antibodies at the times indicated after the addition of cycloheximide, while Vph1-GFP was detected with anti-GFP antibodies. C) Phenylmethylsulfonyl fluoride was added for 1 h toVPH1-GFP cells producing Sna3p-6HA or Sna3p-4KR-6HA before the cells were fixed and processed for immunofluorescence with monoclonal anti-HA and polyclonal anti-GFP antibodies followed by Texas Red-labeled donkey anti-mouse IgG and FITC-labeled donkey anti-rabbit IgG as described in Materials and Methods. Cells were examined for fluorescence (the HA signal was detected using the rhodamine filter set and the GFP signal using the FITC filter set) and vacuole by Nomarski optics. IP: immunoprecipitation, WB: western-blot.
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Related In: Results  -  Collection

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fig05: Sna3p undergoes Ub-dependent MVB sorting.sna3Δand VPH1-GFP cells expressing Sna3p-6HA or Sna3p-4KR-6HA were grown to mid-exponential phase. A) Sna3p-6HA or Sna3p-4KR-6HA produced in sna3Δ cells were detected by immunoblotting of cell lysates with anti-HA antibodies (left panel) and immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies (right panel). B) The production of Sna3p-6HA or Sna3p-4KR-6HA in VPH1-GFP cells was stopped by adding cycloheximide (100 μg/mL). Sna3p-6HA or Sna3p-4KR-6HA was detected by immunoblotting of cell lysates with anti-HA antibodies at the times indicated after the addition of cycloheximide, while Vph1-GFP was detected with anti-GFP antibodies. C) Phenylmethylsulfonyl fluoride was added for 1 h toVPH1-GFP cells producing Sna3p-6HA or Sna3p-4KR-6HA before the cells were fixed and processed for immunofluorescence with monoclonal anti-HA and polyclonal anti-GFP antibodies followed by Texas Red-labeled donkey anti-mouse IgG and FITC-labeled donkey anti-rabbit IgG as described in Materials and Methods. Cells were examined for fluorescence (the HA signal was detected using the rhodamine filter set and the GFP signal using the FITC filter set) and vacuole by Nomarski optics. IP: immunoprecipitation, WB: western-blot.
Mentions: We investigated the role of Sna3p ubiquitylation in its MVB sorting both by biochemical and by immunofluorescence approaches using Sna3p tagged with HA, which does not carry any Lys residue. As immunofluorescent data using Sna3p-HA gave a signal that was too faint, we used a chimeric Sna3p carrying 6HA to improve the signal. We first checked the ubiquitylation fate of the fusion protein. Immunoblot of Sna3Δ cells expressing Sna3p-6HA revealed above the main Sna3p-6HA signal a ladder of bands of higher molecular weight, corresponding to ubiquitylated bands as evidenced by immunoprecipitation with antibodies against HA, followed by immunoblot with anti-Ub antibodies (Figure 5A). Sna3p-6HA ubiquitylation was completely abolished after mutation of four Sna3p lysines on the fusion protein, as revealed by both direct immunoblots and immunoprecipitation using anti-HA antibodies, followed by immunoblot with anti-Ub antibodies (Figure 5A). Hence, Sna3p-6HA mutated or not on its Lys residues was an appropriate tool for our purpose. We analyzed the stability and localization of Sna3-6HA and its four Lys-to-Arg counterpart in cells expressing a GFP-tagged version of Vph1p, a membrane subunit of the vacuolar adenosine triphosphatase (ATPase). We first investigated the turnover of Sna3-6HA and Sna3p-4KR-6HA after inhibiting protein synthesis by adding cycloheximide to exponentially growing cells (Figure 5B). Addition of cycloheximide for 1 h caused apparently no loss of Sna3p-4KR-6HA, while we observed a significant decrease in the amount of Sna3p-6HA compared with the control protein Vph1-GFP. This result indicates that a version of Sna3p that cannot be ubiquitylated is protected against vacuolar degradation. Next, we determined the respective localization of the two proteins by indirect immunofluorescence microscopy. To facilitate localization studies, phenylmethylsulfonyl fluoride (PMSF) was added for 1 h in exponentially grown cells to inhibit vacuolar proteases before cells were fixed and processed for immunofluorescence with anti-HA and anti-GFP antibodies (Figure 5C). Sna3p-6HA was clearly evidenced in vacuole interior, as judged by colocalization of the HA-specific fluorescent signal with vacuoles, detected by Nomarski optics. In contrast, we observed that the HA-specific signal of Sna3p-4KR-6HA co-localized with the GFP-specific signal of the vacuolar membrane marker, Vph1-GFP. Both biochemical and immunofluorescence microscopy data thus strongly suggest that the Lys-to-Arg version of Sna3p is not targeted to the vacuole interior. Therefore, as described for other MVB cargoes, Sna3p ubiquitylation is clearly required for its MVB sorting.

Bottom Line: This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen.Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting.Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS, Universités Paris VI et VII, Département de Biologie Cellulaire, 2 Place Jussieu 75005 Paris, France.

ABSTRACT
Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.

Show MeSH
Related in: MedlinePlus