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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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Targeting of Sna3-GFP to the vacuolar lumen is abolished in rsp5 mutant cells and if the Sna3p PY motif is altered.Microscopic images of Sna3-GFP in living cells. Cells transformed by a Sna3-GFP plasmid under the control of the TPI1 promoter were grown to mid-exponential growth phase and the fluorescence was examined under the microscope. A) Green fluorescent protein and CMAC staining in 27061b parental strain and its derivatives, npi1, tul1Δ and npi1 end3Δ mutant cells; in the BG1 parental strain and derivative cells deleted for RSP5 and producing a centromeric plasmid version of Rsp5p wild type (WT) or mutated in the WW1, WW2 or WW3 domain; and in BY parental strain, pep12Δ and bul1Δbul2Δ mutant cells. The identity of the vacuole was confirmed by staining with the dye CMAC as described in Materials and Methods. B) Western blots from total protein extracts from WT and npi1 mutant cells producing Sna3-GFP were probed with anti-GFP antibodies. The sizes of molecular weight markers are indicated. C) Cells transformed by a plasmid encoding GFP-tagged WT or Sna3p variants were grown to mid-exponential phase and cells were examined for fluorescence and with Nomarski (Nom) optics. *, the image of Sna3-AY-GFP is magnified.
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fig02: Targeting of Sna3-GFP to the vacuolar lumen is abolished in rsp5 mutant cells and if the Sna3p PY motif is altered.Microscopic images of Sna3-GFP in living cells. Cells transformed by a Sna3-GFP plasmid under the control of the TPI1 promoter were grown to mid-exponential growth phase and the fluorescence was examined under the microscope. A) Green fluorescent protein and CMAC staining in 27061b parental strain and its derivatives, npi1, tul1Δ and npi1 end3Δ mutant cells; in the BG1 parental strain and derivative cells deleted for RSP5 and producing a centromeric plasmid version of Rsp5p wild type (WT) or mutated in the WW1, WW2 or WW3 domain; and in BY parental strain, pep12Δ and bul1Δbul2Δ mutant cells. The identity of the vacuole was confirmed by staining with the dye CMAC as described in Materials and Methods. B) Western blots from total protein extracts from WT and npi1 mutant cells producing Sna3-GFP were probed with anti-GFP antibodies. The sizes of molecular weight markers are indicated. C) Cells transformed by a plasmid encoding GFP-tagged WT or Sna3p variants were grown to mid-exponential phase and cells were examined for fluorescence and with Nomarski (Nom) optics. *, the image of Sna3-AY-GFP is magnified.

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)

Targeting of Sna3-GFP to the vacuolar lumen is abolished in rsp5 mutant cells and if the Sna3p PY motif is altered.Microscopic images of Sna3-GFP in living cells. Cells transformed by a Sna3-GFP plasmid under the control of the TPI1 promoter were grown to mid-exponential growth phase and the fluorescence was examined under the microscope. A) Green fluorescent protein and CMAC staining in 27061b parental strain and its derivatives, npi1, tul1Δ and npi1 end3Δ mutant cells; in the BG1 parental strain and derivative cells deleted for RSP5 and producing a centromeric plasmid version of Rsp5p wild type (WT) or mutated in the WW1, WW2 or WW3 domain; and in BY parental strain, pep12Δ and bul1Δbul2Δ mutant cells. The identity of the vacuole was confirmed by staining with the dye CMAC as described in Materials and Methods. B) Western blots from total protein extracts from WT and npi1 mutant cells producing Sna3-GFP were probed with anti-GFP antibodies. The sizes of molecular weight markers are indicated. C) Cells transformed by a plasmid encoding GFP-tagged WT or Sna3p variants were grown to mid-exponential phase and cells were examined for fluorescence and with Nomarski (Nom) optics. *, the image of Sna3-AY-GFP is magnified.
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Related In: Results  -  Collection

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fig02: Targeting of Sna3-GFP to the vacuolar lumen is abolished in rsp5 mutant cells and if the Sna3p PY motif is altered.Microscopic images of Sna3-GFP in living cells. Cells transformed by a Sna3-GFP plasmid under the control of the TPI1 promoter were grown to mid-exponential growth phase and the fluorescence was examined under the microscope. A) Green fluorescent protein and CMAC staining in 27061b parental strain and its derivatives, npi1, tul1Δ and npi1 end3Δ mutant cells; in the BG1 parental strain and derivative cells deleted for RSP5 and producing a centromeric plasmid version of Rsp5p wild type (WT) or mutated in the WW1, WW2 or WW3 domain; and in BY parental strain, pep12Δ and bul1Δbul2Δ mutant cells. The identity of the vacuole was confirmed by staining with the dye CMAC as described in Materials and Methods. B) Western blots from total protein extracts from WT and npi1 mutant cells producing Sna3-GFP were probed with anti-GFP antibodies. The sizes of molecular weight markers are indicated. C) Cells transformed by a plasmid encoding GFP-tagged WT or Sna3p variants were grown to mid-exponential phase and cells were examined for fluorescence and with Nomarski (Nom) optics. *, the image of Sna3-AY-GFP is magnified.
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