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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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Binding of the PY motif of Sna3p to the WW domains of Rsp5p.A) Schematic representation of Rsp5p and Sna3p. B) Immunoprecipitation was performed with 170 μg of total extracts from cells producing HA-tagged Rsp5p under the control of its own promoter and transformed by a plasmid encoding GFP-tagged Sna3p wild type or mutants under the control of TPI1 promoter. Tagged proteins were immunoprecipitated with anti-HA or anti-GFP antibodies. Cells producing untagged Rsp5p or Sna3p were used as controls. Immunoprecipitated proteins were detected by immunoblotting with anti-HA and anti-GFP antibodies. Tot: 6% of the solubilized proteins were loaded on the gel. IP: 50% of the immunoprecipitated material was loaded on the gel. C) Glutathione S-transferase and GST-WW2/WW3 Rsp5p domain recombinant proteins bound to glutathione–Sepharose beads were incubated with extracts from cells producing Sna3-GFP, Sna3-AY-GFP or GFP-Cps1p. Total extract (T), unbound (UnB) and bound (B) fractions were analyzed by Western blotting with anti-GFP. °: These bands were demonstrated to be ubiquitylated Sna3p species (see later). IP: immunoprecipitation, WB: western-blot.
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fig01: Binding of the PY motif of Sna3p to the WW domains of Rsp5p.A) Schematic representation of Rsp5p and Sna3p. B) Immunoprecipitation was performed with 170 μg of total extracts from cells producing HA-tagged Rsp5p under the control of its own promoter and transformed by a plasmid encoding GFP-tagged Sna3p wild type or mutants under the control of TPI1 promoter. Tagged proteins were immunoprecipitated with anti-HA or anti-GFP antibodies. Cells producing untagged Rsp5p or Sna3p were used as controls. Immunoprecipitated proteins were detected by immunoblotting with anti-HA and anti-GFP antibodies. Tot: 6% of the solubilized proteins were loaded on the gel. IP: 50% of the immunoprecipitated material was loaded on the gel. C) Glutathione S-transferase and GST-WW2/WW3 Rsp5p domain recombinant proteins bound to glutathione–Sepharose beads were incubated with extracts from cells producing Sna3-GFP, Sna3-AY-GFP or GFP-Cps1p. Total extract (T), unbound (UnB) and bound (B) fractions were analyzed by Western blotting with anti-GFP. °: These bands were demonstrated to be ubiquitylated Sna3p species (see later). IP: immunoprecipitation, WB: western-blot.

Mentions: Rsp5p is a modular protein with an N-terminal C2 domain that may interact with membranes (by binding to lipids or membrane proteins), three tryptophan-rich WW domains involved in protein–protein interactions with proline-rich peptides and a C-terminal catalytic HECT domain (Figure 1A). There is now a considerable body of data to suggest that the tryptophan-rich WW domains of the Nedd4 family members, or a subset of these domains, are involved, either directly or indirectly, in substrate recognition. Based on the presence of signature residues, a classification scheme has been proposed for WW domains (19). The three WW domains of Rsp5p belong to the group I domains that mediate protein–protein interactions via the PPXY (PY) motifs (20). The biosynthetic vacuolar protein Sna3p possesses a PY motif (sequence PPAY) in its C-terminus cytoplasmic part (Figure 1A). This prompted us to investigate the possibility that Sna3p interacts with the Ub ligase Rsp5p via its PPAY sequence. We conducted co-immunoprecipitation experiments with cells expressing both influenza hemagglutinin protein (HA)-tagged Rsp5p under the control of its own promoter and GFP-tagged Sna3p under the control of the TPI1 promoter (Figure 1B). First, we performed immunoprecipitations with antibodies recognizing HA and then probed immunoprecipitated proteins with HA or GFP antibodies. Figure 1B shows that Sna3-GFP was efficiently co-immunoprecipitated with HA-Rsp5p. Fifteen percent of the solubilized HA-Rsp5p was recovered after immunoprecipitation, and 4% of the solubilized Sna3-GFP was co-immunoprecipitated. In contrast, Sna3-GFP was not detected in the control experiment, which was performed on cells producing untagged Rsp5p. Immunoprecipitations were also performed with antibodies recognizing GFP. Immunoprecipitated proteins were probed with HA or GFP antibodies revealing the presence of both Rsp5p-HA and Sna3p-GFP after immunoprecipitation. Fifteen percent of the solubilized HA-Rsp5p was recovered after immunoprecipitation, and 4% of the solubilized Sna3-GFP was co-immunoprecipitated. Rsp5p-HA was not detected in the control experiment, which was performed on cells expressing untagged Sna3p. These results indicate that Sna3p interacts either directly or indirectly with Rsp5p. To determine whether the Sna3p PPAY sequence is required for Rsp5p binding, we performed co-immunoprecipitation assays from HA-tagged Rsp5p-expressing cells transformed by a plasmid encoding a mutant form of Sna3-GFP in which the PY motif was deleted (mutant Δ28) or modified for AAAY (mutant AY). As shown in Figure 1B, these two variant forms that lack the PY motif were unable to interact with Rsp5p-HA, confirming the importance of the PY motif for this interaction and supporting the idea of a direct interaction.


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)

Binding of the PY motif of Sna3p to the WW domains of Rsp5p.A) Schematic representation of Rsp5p and Sna3p. B) Immunoprecipitation was performed with 170 μg of total extracts from cells producing HA-tagged Rsp5p under the control of its own promoter and transformed by a plasmid encoding GFP-tagged Sna3p wild type or mutants under the control of TPI1 promoter. Tagged proteins were immunoprecipitated with anti-HA or anti-GFP antibodies. Cells producing untagged Rsp5p or Sna3p were used as controls. Immunoprecipitated proteins were detected by immunoblotting with anti-HA and anti-GFP antibodies. Tot: 6% of the solubilized proteins were loaded on the gel. IP: 50% of the immunoprecipitated material was loaded on the gel. C) Glutathione S-transferase and GST-WW2/WW3 Rsp5p domain recombinant proteins bound to glutathione–Sepharose beads were incubated with extracts from cells producing Sna3-GFP, Sna3-AY-GFP or GFP-Cps1p. Total extract (T), unbound (UnB) and bound (B) fractions were analyzed by Western blotting with anti-GFP. °: These bands were demonstrated to be ubiquitylated Sna3p species (see later). IP: immunoprecipitation, WB: western-blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171029&req=5

fig01: Binding of the PY motif of Sna3p to the WW domains of Rsp5p.A) Schematic representation of Rsp5p and Sna3p. B) Immunoprecipitation was performed with 170 μg of total extracts from cells producing HA-tagged Rsp5p under the control of its own promoter and transformed by a plasmid encoding GFP-tagged Sna3p wild type or mutants under the control of TPI1 promoter. Tagged proteins were immunoprecipitated with anti-HA or anti-GFP antibodies. Cells producing untagged Rsp5p or Sna3p were used as controls. Immunoprecipitated proteins were detected by immunoblotting with anti-HA and anti-GFP antibodies. Tot: 6% of the solubilized proteins were loaded on the gel. IP: 50% of the immunoprecipitated material was loaded on the gel. C) Glutathione S-transferase and GST-WW2/WW3 Rsp5p domain recombinant proteins bound to glutathione–Sepharose beads were incubated with extracts from cells producing Sna3-GFP, Sna3-AY-GFP or GFP-Cps1p. Total extract (T), unbound (UnB) and bound (B) fractions were analyzed by Western blotting with anti-GFP. °: These bands were demonstrated to be ubiquitylated Sna3p species (see later). IP: immunoprecipitation, WB: western-blot.
Mentions: Rsp5p is a modular protein with an N-terminal C2 domain that may interact with membranes (by binding to lipids or membrane proteins), three tryptophan-rich WW domains involved in protein–protein interactions with proline-rich peptides and a C-terminal catalytic HECT domain (Figure 1A). There is now a considerable body of data to suggest that the tryptophan-rich WW domains of the Nedd4 family members, or a subset of these domains, are involved, either directly or indirectly, in substrate recognition. Based on the presence of signature residues, a classification scheme has been proposed for WW domains (19). The three WW domains of Rsp5p belong to the group I domains that mediate protein–protein interactions via the PPXY (PY) motifs (20). The biosynthetic vacuolar protein Sna3p possesses a PY motif (sequence PPAY) in its C-terminus cytoplasmic part (Figure 1A). This prompted us to investigate the possibility that Sna3p interacts with the Ub ligase Rsp5p via its PPAY sequence. We conducted co-immunoprecipitation experiments with cells expressing both influenza hemagglutinin protein (HA)-tagged Rsp5p under the control of its own promoter and GFP-tagged Sna3p under the control of the TPI1 promoter (Figure 1B). First, we performed immunoprecipitations with antibodies recognizing HA and then probed immunoprecipitated proteins with HA or GFP antibodies. Figure 1B shows that Sna3-GFP was efficiently co-immunoprecipitated with HA-Rsp5p. Fifteen percent of the solubilized HA-Rsp5p was recovered after immunoprecipitation, and 4% of the solubilized Sna3-GFP was co-immunoprecipitated. In contrast, Sna3-GFP was not detected in the control experiment, which was performed on cells producing untagged Rsp5p. Immunoprecipitations were also performed with antibodies recognizing GFP. Immunoprecipitated proteins were probed with HA or GFP antibodies revealing the presence of both Rsp5p-HA and Sna3p-GFP after immunoprecipitation. Fifteen percent of the solubilized HA-Rsp5p was recovered after immunoprecipitation, and 4% of the solubilized Sna3-GFP was co-immunoprecipitated. Rsp5p-HA was not detected in the control experiment, which was performed on cells expressing untagged Sna3p. These results indicate that Sna3p interacts either directly or indirectly with Rsp5p. To determine whether the Sna3p PPAY sequence is required for Rsp5p binding, we performed co-immunoprecipitation assays from HA-tagged Rsp5p-expressing cells transformed by a plasmid encoding a mutant form of Sna3-GFP in which the PY motif was deleted (mutant Δ28) or modified for AAAY (mutant AY). As shown in Figure 1B, these two variant forms that lack the PY motif were unable to interact with Rsp5p-HA, confirming the importance of the PY motif for this interaction and supporting the idea of a direct interaction.

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