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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 is polyubiquitylated on one target lysine, K125.A) Cells expressing Sna3-K19,125R-GFP and Sna3-4KR-GFP were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski (Nom) optics. B) doa4Δ, vps23Δ, vps36Δ and vps24Δ cells transformed by a Sna3-GFP encoding plasmid were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski optics. C) Left panel: HA-tagged version of Sna3p and Sna3-K125R were detected by immunoblotting of corresponding cell lysates from wild-type (WT) cells with anti-HA antibodies. Right panel: Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies. D) Modified forms of Sna3-GFP and its KR mutant derivatives were detected by immunoblotting with anti-GFP antibodies or by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-GFP. The sizes of molecular weight markers are indicated. IP: immunoprecipitation, WB: western-blot.
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fig04: Sna3p is polyubiquitylated on one target lysine, K125.A) Cells expressing Sna3-K19,125R-GFP and Sna3-4KR-GFP were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski (Nom) optics. B) doa4Δ, vps23Δ, vps36Δ and vps24Δ cells transformed by a Sna3-GFP encoding plasmid were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski optics. C) Left panel: HA-tagged version of Sna3p and Sna3-K125R were detected by immunoblotting of corresponding cell lysates from wild-type (WT) cells with anti-HA antibodies. Right panel: Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies. D) Modified forms of Sna3-GFP and its KR mutant derivatives were detected by immunoblotting with anti-GFP antibodies or by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-GFP. The sizes of molecular weight markers are indicated. IP: immunoprecipitation, WB: western-blot.

Mentions: We expressed GFP-tagged Sna3p variants in which the four lysine residues of Sna3p (see Figure 1A) were replaced by Arg in various combinations, hence making ubiquitylation of these residues impossible, and we indeed observed a lumenal localization of Sna3-K19,125R–GFP and Sna3-4KR–GFP in cells otherwise deleted (Figure 4A) or not (data not shown) for the chromosomal copy of Sna3p. We thus eliminated the possibility that sorting of the mutant Sna3p occurs as a result of oligomerization with endogenous wild-type Sna3p. These data confirm the results obtained by Reggiori and Pelham (10). Sna3-GFP is targeted to the vacuolar lumen without being modified by Ub added to the Sna3 moiety of the chimeric protein. We further tested whether Sna3-GFP used all or part of the ESCRT machinery required for MVB sorting of ubiquitylated membrane cargoes. We expressed wild-type Sna3-GFP from the inducible GAL promoter in mutant cells deleted for each of the subunits of the ESCRT machinery. The Sna3-GFP fluorescence in vps23Δ/Tsg101 (ESCRT-I), vps36Δ (ESCRT-II) and vps24Δ (ESCRT-III) mutant cells is shown in Figure 4B. Vps23p and Vps36p both possess a Ub-binding motif that recognizes ubiquitylated cargoes (3). In all cases, Sna3-GFP stained one or two big dots, adjacent to the vacuole, which might represent the class E compartment. Our data suggest that all the elements of the ESCRT machinery are required for Sna3-GFP sorting at the MVB. Strikingly, in our genetic background, in addition to the localization in the vacuolar lumen of some cells, a class E-type fluorescence was observed for Sna3-GFP expressed in doa4Δ cells (Figure 4B). A more systematic investigation of Sna3-GFP fate in doa4Δ cells from different genetic backgrounds showed more or less normal sorting in cells in early exponential phase, notably in the genetic background used by Reggiori and Pelham (10) with increasing defects at higher cell densities (i.e. late exponential growth phase) (data not shown), in agreement with the observation of decreased Ub content in doa4Δ cells along cell growth (31).


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 is polyubiquitylated on one target lysine, K125.A) Cells expressing Sna3-K19,125R-GFP and Sna3-4KR-GFP were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski (Nom) optics. B) doa4Δ, vps23Δ, vps36Δ and vps24Δ cells transformed by a Sna3-GFP encoding plasmid were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski optics. C) Left panel: HA-tagged version of Sna3p and Sna3-K125R were detected by immunoblotting of corresponding cell lysates from wild-type (WT) cells with anti-HA antibodies. Right panel: Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies. D) Modified forms of Sna3-GFP and its KR mutant derivatives were detected by immunoblotting with anti-GFP antibodies or by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-GFP. The sizes of molecular weight markers are indicated. IP: immunoprecipitation, WB: western-blot.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171029&req=5

fig04: Sna3p is polyubiquitylated on one target lysine, K125.A) Cells expressing Sna3-K19,125R-GFP and Sna3-4KR-GFP were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski (Nom) optics. B) doa4Δ, vps23Δ, vps36Δ and vps24Δ cells transformed by a Sna3-GFP encoding plasmid were grown to mid-exponential growth phase, and cells were examined for fluorescence and with Nomarski optics. C) Left panel: HA-tagged version of Sna3p and Sna3-K125R were detected by immunoblotting of corresponding cell lysates from wild-type (WT) cells with anti-HA antibodies. Right panel: Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA antibodies. D) Modified forms of Sna3-GFP and its KR mutant derivatives were detected by immunoblotting with anti-GFP antibodies or by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-GFP. The sizes of molecular weight markers are indicated. IP: immunoprecipitation, WB: western-blot.
Mentions: We expressed GFP-tagged Sna3p variants in which the four lysine residues of Sna3p (see Figure 1A) were replaced by Arg in various combinations, hence making ubiquitylation of these residues impossible, and we indeed observed a lumenal localization of Sna3-K19,125R–GFP and Sna3-4KR–GFP in cells otherwise deleted (Figure 4A) or not (data not shown) for the chromosomal copy of Sna3p. We thus eliminated the possibility that sorting of the mutant Sna3p occurs as a result of oligomerization with endogenous wild-type Sna3p. These data confirm the results obtained by Reggiori and Pelham (10). Sna3-GFP is targeted to the vacuolar lumen without being modified by Ub added to the Sna3 moiety of the chimeric protein. We further tested whether Sna3-GFP used all or part of the ESCRT machinery required for MVB sorting of ubiquitylated membrane cargoes. We expressed wild-type Sna3-GFP from the inducible GAL promoter in mutant cells deleted for each of the subunits of the ESCRT machinery. The Sna3-GFP fluorescence in vps23Δ/Tsg101 (ESCRT-I), vps36Δ (ESCRT-II) and vps24Δ (ESCRT-III) mutant cells is shown in Figure 4B. Vps23p and Vps36p both possess a Ub-binding motif that recognizes ubiquitylated cargoes (3). In all cases, Sna3-GFP stained one or two big dots, adjacent to the vacuole, which might represent the class E compartment. Our data suggest that all the elements of the ESCRT machinery are required for Sna3-GFP sorting at the MVB. Strikingly, in our genetic background, in addition to the localization in the vacuolar lumen of some cells, a class E-type fluorescence was observed for Sna3-GFP expressed in doa4Δ cells (Figure 4B). A more systematic investigation of Sna3-GFP fate in doa4Δ cells from different genetic backgrounds showed more or less normal sorting in cells in early exponential phase, notably in the genetic background used by Reggiori and Pelham (10) with increasing defects at higher cell densities (i.e. late exponential growth phase) (data not shown), in agreement with the observation of decreased Ub content in doa4Δ cells along cell growth (31).

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