Limits...
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.

Show MeSH

Related in: MedlinePlus

Sna3p is ubiquitylated by the E3 ligase Rsp5p.A) Green fluorescent protein-tagged version of Sna3p was detected either by immunoblotting of a cell lysate from wild-type (WT) cells with anti-GFP or by immunoblotting with anti-Ub or anti-GFP after immunoprecipitation with anti-GFP. Molecular weight markers sizes are indicated. B) Rsp5p and PGK, a loading control protein, were detected by immunoblotting of a cell lysate from doa4Δ and doa4Δnpi1 cells with anti-mNedd4 and anti-PGK antibodies, respectively. C) doa4Δ, doa4Δnpi1 and doa4Δtul1Δ cells co-producing Sna3-GFP and CUP1 promoter-driven 6His-Ub were grown to mid-exponential phase. CuSO4 (100 μm) was added, and the cells were incubated for an additional 3 h to induce the CUP1 promoter. Extracts of 6 × 108 to 7 × 108 cells were fractionated as described in Materials and Methods. All experiments were conducted in identical conditions of growth and cell fractionation in at least three independent experiments. Solubilized membranes were passed through Ni-NTA columns. The unbound fractions were collected and the 6His-tagged ubiquitylated proteins were then eluted using a buffer containing 200 mm imidazole. Aliquots of each fraction were resolved by electrophoresis and analyzed by Western immunoblotting with anti-GFP antibodies. Solubilized membrane fractions (S), unbound fraction (UnB) and purified 6His-tagged proteins (B) are shown. The lane on the right is overexposed. Arrowheads indicate Sna3-GFP conjugated to 6His-tagged Ub moieties. IP: immunoprecipitation, WB: western-blot.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171029&req=5

fig03: Sna3p is ubiquitylated by the E3 ligase Rsp5p.A) Green fluorescent protein-tagged version of Sna3p was detected either by immunoblotting of a cell lysate from wild-type (WT) cells with anti-GFP or by immunoblotting with anti-Ub or anti-GFP after immunoprecipitation with anti-GFP. Molecular weight markers sizes are indicated. B) Rsp5p and PGK, a loading control protein, were detected by immunoblotting of a cell lysate from doa4Δ and doa4Δnpi1 cells with anti-mNedd4 and anti-PGK antibodies, respectively. C) doa4Δ, doa4Δnpi1 and doa4Δtul1Δ cells co-producing Sna3-GFP and CUP1 promoter-driven 6His-Ub were grown to mid-exponential phase. CuSO4 (100 μm) was added, and the cells were incubated for an additional 3 h to induce the CUP1 promoter. Extracts of 6 × 108 to 7 × 108 cells were fractionated as described in Materials and Methods. All experiments were conducted in identical conditions of growth and cell fractionation in at least three independent experiments. Solubilized membranes were passed through Ni-NTA columns. The unbound fractions were collected and the 6His-tagged ubiquitylated proteins were then eluted using a buffer containing 200 mm imidazole. Aliquots of each fraction were resolved by electrophoresis and analyzed by Western immunoblotting with anti-GFP antibodies. Solubilized membrane fractions (S), unbound fraction (UnB) and purified 6His-tagged proteins (B) are shown. The lane on the right is overexposed. Arrowheads indicate Sna3-GFP conjugated to 6His-tagged Ub moieties. IP: immunoprecipitation, WB: western-blot.

Mentions: We then investigated the potential role of this Rsp5p interaction in the sorting of Sna3p by analyzing the subcellular distribution of Sna3p fused to the GFP in various rsp5 mutant cells relative to wild-type cells. When expressed in wild-type cells of various genetic backgrounds, Sna3-GFP was delivered to the lumen of the vacuole, resulting in GFP fluorescence in the vacuole interior, as shown by the identical localization obtained with the vacuolar cell tracker blue CMAC dye (Figure 2A). We then investigated the role played by Rsp5p in the delivery of Sna3-GFP to the vacuole (Figure 2A). We first used viable npi1 mutant cells, npi1 being a mutant allele of RSP5 with a reduced expression of Rsp5p because of the insertion of a Ty element in the RSP5 promoter (25,26). Using antibodies against mNedd4, the mouse homologue of Rsp5p, we were able to confirm that this mutant produced very small amounts of Rsp5p (Figure 3B). In npi1 mutant cells, Sna3-GFP accumulated in very small and mobile structures, probably vesicles (Figure 2A). These diffuse stained dots were also observed in temperature-sensitive rsp5-101ts mutant cells shifted to nonpermissive temperature (data not shown). Sna3-GFP delocalization was identical in npi1 end3Δ cells, also defective for the internalization step of endocytosis, indicating that Sna3-GFP was sorted directly from the Golgi to these small vesicles rather than via the plasma membrane followed by subsequent endocytosis. This vesicular pattern is similar to that observed upon expression of Sna3-GFP in pep12Δ, defective for the fusion of vesicles with the late endosome.


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 ubiquitylated by the E3 ligase Rsp5p.A) Green fluorescent protein-tagged version of Sna3p was detected either by immunoblotting of a cell lysate from wild-type (WT) cells with anti-GFP or by immunoblotting with anti-Ub or anti-GFP after immunoprecipitation with anti-GFP. Molecular weight markers sizes are indicated. B) Rsp5p and PGK, a loading control protein, were detected by immunoblotting of a cell lysate from doa4Δ and doa4Δnpi1 cells with anti-mNedd4 and anti-PGK antibodies, respectively. C) doa4Δ, doa4Δnpi1 and doa4Δtul1Δ cells co-producing Sna3-GFP and CUP1 promoter-driven 6His-Ub were grown to mid-exponential phase. CuSO4 (100 μm) was added, and the cells were incubated for an additional 3 h to induce the CUP1 promoter. Extracts of 6 × 108 to 7 × 108 cells were fractionated as described in Materials and Methods. All experiments were conducted in identical conditions of growth and cell fractionation in at least three independent experiments. Solubilized membranes were passed through Ni-NTA columns. The unbound fractions were collected and the 6His-tagged ubiquitylated proteins were then eluted using a buffer containing 200 mm imidazole. Aliquots of each fraction were resolved by electrophoresis and analyzed by Western immunoblotting with anti-GFP antibodies. Solubilized membrane fractions (S), unbound fraction (UnB) and purified 6His-tagged proteins (B) are shown. The lane on the right is overexposed. Arrowheads indicate Sna3-GFP conjugated to 6His-tagged Ub moieties. IP: immunoprecipitation, WB: western-blot.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Sna3p is ubiquitylated by the E3 ligase Rsp5p.A) Green fluorescent protein-tagged version of Sna3p was detected either by immunoblotting of a cell lysate from wild-type (WT) cells with anti-GFP or by immunoblotting with anti-Ub or anti-GFP after immunoprecipitation with anti-GFP. Molecular weight markers sizes are indicated. B) Rsp5p and PGK, a loading control protein, were detected by immunoblotting of a cell lysate from doa4Δ and doa4Δnpi1 cells with anti-mNedd4 and anti-PGK antibodies, respectively. C) doa4Δ, doa4Δnpi1 and doa4Δtul1Δ cells co-producing Sna3-GFP and CUP1 promoter-driven 6His-Ub were grown to mid-exponential phase. CuSO4 (100 μm) was added, and the cells were incubated for an additional 3 h to induce the CUP1 promoter. Extracts of 6 × 108 to 7 × 108 cells were fractionated as described in Materials and Methods. All experiments were conducted in identical conditions of growth and cell fractionation in at least three independent experiments. Solubilized membranes were passed through Ni-NTA columns. The unbound fractions were collected and the 6His-tagged ubiquitylated proteins were then eluted using a buffer containing 200 mm imidazole. Aliquots of each fraction were resolved by electrophoresis and analyzed by Western immunoblotting with anti-GFP antibodies. Solubilized membrane fractions (S), unbound fraction (UnB) and purified 6His-tagged proteins (B) are shown. The lane on the right is overexposed. Arrowheads indicate Sna3-GFP conjugated to 6His-tagged Ub moieties. IP: immunoprecipitation, WB: western-blot.
Mentions: We then investigated the potential role of this Rsp5p interaction in the sorting of Sna3p by analyzing the subcellular distribution of Sna3p fused to the GFP in various rsp5 mutant cells relative to wild-type cells. When expressed in wild-type cells of various genetic backgrounds, Sna3-GFP was delivered to the lumen of the vacuole, resulting in GFP fluorescence in the vacuole interior, as shown by the identical localization obtained with the vacuolar cell tracker blue CMAC dye (Figure 2A). We then investigated the role played by Rsp5p in the delivery of Sna3-GFP to the vacuole (Figure 2A). We first used viable npi1 mutant cells, npi1 being a mutant allele of RSP5 with a reduced expression of Rsp5p because of the insertion of a Ty element in the RSP5 promoter (25,26). Using antibodies against mNedd4, the mouse homologue of Rsp5p, we were able to confirm that this mutant produced very small amounts of Rsp5p (Figure 3B). In npi1 mutant cells, Sna3-GFP accumulated in very small and mobile structures, probably vesicles (Figure 2A). These diffuse stained dots were also observed in temperature-sensitive rsp5-101ts mutant cells shifted to nonpermissive temperature (data not shown). Sna3-GFP delocalization was identical in npi1 end3Δ cells, also defective for the internalization step of endocytosis, indicating that Sna3-GFP was sorted directly from the Golgi to these small vesicles rather than via the plasma membrane followed by subsequent endocytosis. This vesicular pattern is similar to that observed upon expression of Sna3-GFP in pep12Δ, defective for the fusion of vesicles with the late endosome.

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