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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 modified by Lys63-linked Ub chains.A) doa4Δ cells co-transformed with Sna3p-HA encoding plasmid and plasmids encoding wild-type (wt) Ub, UbK29R, UbK48R or UbK63R were grown to mid-exponential phase and induced for Ub synthesis for 1 h as described in Figure 3C. Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The positions of size markers are indicated. B) Sna3-HA ubiquitylation in cells whose sole source of Ub was a modified Ub. SUB280 and derivative strains producing wild-type Ub or a modified form of Ub, respectively, were transformed Sna3p-HA encoding plasmid. Cells were grown to mid-exponential phase and modified forms of Sna3-HA were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The sizes of molecular weight markers are indicated.
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fig06: Sna3p is modified by Lys63-linked Ub chains.A) doa4Δ cells co-transformed with Sna3p-HA encoding plasmid and plasmids encoding wild-type (wt) Ub, UbK29R, UbK48R or UbK63R were grown to mid-exponential phase and induced for Ub synthesis for 1 h as described in Figure 3C. Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The positions of size markers are indicated. B) Sna3-HA ubiquitylation in cells whose sole source of Ub was a modified Ub. SUB280 and derivative strains producing wild-type Ub or a modified form of Ub, respectively, were transformed Sna3p-HA encoding plasmid. Cells were grown to mid-exponential phase and modified forms of Sna3-HA were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The sizes of molecular weight markers are indicated.

Mentions: Little is as yet known about the ubiquitylation features associated with Golgi to vacuole trafficking. Some transporters misrouted from Golgi to vacuole are thought to undergo Rsp5-dependent polyubiquitylation, but the types of Ub chain have not been characterized (4,6,33). In contrast, MVB cargoes are thought to be primarily monoubiquitylated (30). We attempted to characterize ubiquitylation associated with Sna3p trafficking. Complementing Ub levels in doa4Δ cells with plasmid-encoded Ub provides a powerful tool for characterizing the types of Ub chain carried by target proteins (34,35). We therefore used doa4Δ cells to test the effect of various mutated Ubs on Sna3p-HA ubiquitylation. doa4Δ cells were grown to mid-exponential growth phase and the production of Ub variants was induced by adding copper salt for 1 h before harvest. By overexpressing wild-type Ub in doa4Δ cells, more than eight Ub conjugates were evidenced when Sna3p-HA was immunoprecipitated with anti-HA and Ub conjugates were revealed with anti-Ub antibodies (Figure 6A). The pattern of ubiquitylation was similar to that previously observed in wild-type cells (Figure 4C). Overexpression of UbK29R and UbK48R in doa4Δ cells also restored a normal ubiquitylation pattern of Sna3p-HA. In contrast, after overexpression of UbK63R, only mono- and di-ubiquitylated forms of Sna3p-HA were observed, together with some minor upper bands. These data demonstrated the inability of UbK63R to induce complete polyubiquitylation of Sna3p-HA. However, because Sna3p is ubiquitylated on a unique residue, Lys125, one would expect to observe only a monoubiquitylation of Sna3p-HA in cells overexpressing UbK63R. doa4Δ still produces some endogenous Ub. This wild-type Ub might compete for incorporation in Ub chains and could account for the presence of more than one added residue on Sna3p. In order to ascertain that the Ub profile observed upon overexpression of UbK63R did not result from the pool of chromosomal-encoded Ub, we investigated another experimental system. We analyzed the fate of Sna3p-HA in a strain in which the four natural Ub genes had been disrupted (36) and which expresses plasmid-encoded wild-type Ub or various Lys-to-Arg mutated Ub genes as their sole source of Ub (Figure 6B). The profiles of Sna3p–Ub conjugates were identical in cells producing wild-type Ub or mutated Ub with substitutions at Lys6, 11, 27, 29 and 33. The effect of substituting Lys48 was not tested because it is lethal for the cells (36). In contrast, the pattern of Ub–Sna3p conjugates in cells producing UbK63R as sole source of Ub was similar to that observed in doa4Δ cells overexpressing UbK63R, with the number of Ub conjugates notably reduced. The presence of more than one Ub-conjugated band still remains to be understood, but it is possible that when Lys63-Ub chains cannot be formed, an alternative chain is synthesized, at least in this case. Whatever the case, the overall data clearly indicate that Sna3p is modified by Lys63-Ub chains.


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 modified by Lys63-linked Ub chains.A) doa4Δ cells co-transformed with Sna3p-HA encoding plasmid and plasmids encoding wild-type (wt) Ub, UbK29R, UbK48R or UbK63R were grown to mid-exponential phase and induced for Ub synthesis for 1 h as described in Figure 3C. Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The positions of size markers are indicated. B) Sna3-HA ubiquitylation in cells whose sole source of Ub was a modified Ub. SUB280 and derivative strains producing wild-type Ub or a modified form of Ub, respectively, were transformed Sna3p-HA encoding plasmid. Cells were grown to mid-exponential phase and modified forms of Sna3-HA were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The sizes of molecular weight markers are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171029&req=5

fig06: Sna3p is modified by Lys63-linked Ub chains.A) doa4Δ cells co-transformed with Sna3p-HA encoding plasmid and plasmids encoding wild-type (wt) Ub, UbK29R, UbK48R or UbK63R were grown to mid-exponential phase and induced for Ub synthesis for 1 h as described in Figure 3C. Modified forms of Sna3-HA and its KR mutant derivatives were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The positions of size markers are indicated. B) Sna3-HA ubiquitylation in cells whose sole source of Ub was a modified Ub. SUB280 and derivative strains producing wild-type Ub or a modified form of Ub, respectively, were transformed Sna3p-HA encoding plasmid. Cells were grown to mid-exponential phase and modified forms of Sna3-HA were detected by immunoblotting with anti-Ub antibodies after immunoprecipitation with anti-HA. The sizes of molecular weight markers are indicated.
Mentions: Little is as yet known about the ubiquitylation features associated with Golgi to vacuole trafficking. Some transporters misrouted from Golgi to vacuole are thought to undergo Rsp5-dependent polyubiquitylation, but the types of Ub chain have not been characterized (4,6,33). In contrast, MVB cargoes are thought to be primarily monoubiquitylated (30). We attempted to characterize ubiquitylation associated with Sna3p trafficking. Complementing Ub levels in doa4Δ cells with plasmid-encoded Ub provides a powerful tool for characterizing the types of Ub chain carried by target proteins (34,35). We therefore used doa4Δ cells to test the effect of various mutated Ubs on Sna3p-HA ubiquitylation. doa4Δ cells were grown to mid-exponential growth phase and the production of Ub variants was induced by adding copper salt for 1 h before harvest. By overexpressing wild-type Ub in doa4Δ cells, more than eight Ub conjugates were evidenced when Sna3p-HA was immunoprecipitated with anti-HA and Ub conjugates were revealed with anti-Ub antibodies (Figure 6A). The pattern of ubiquitylation was similar to that previously observed in wild-type cells (Figure 4C). Overexpression of UbK29R and UbK48R in doa4Δ cells also restored a normal ubiquitylation pattern of Sna3p-HA. In contrast, after overexpression of UbK63R, only mono- and di-ubiquitylated forms of Sna3p-HA were observed, together with some minor upper bands. These data demonstrated the inability of UbK63R to induce complete polyubiquitylation of Sna3p-HA. However, because Sna3p is ubiquitylated on a unique residue, Lys125, one would expect to observe only a monoubiquitylation of Sna3p-HA in cells overexpressing UbK63R. doa4Δ still produces some endogenous Ub. This wild-type Ub might compete for incorporation in Ub chains and could account for the presence of more than one added residue on Sna3p. In order to ascertain that the Ub profile observed upon overexpression of UbK63R did not result from the pool of chromosomal-encoded Ub, we investigated another experimental system. We analyzed the fate of Sna3p-HA in a strain in which the four natural Ub genes had been disrupted (36) and which expresses plasmid-encoded wild-type Ub or various Lys-to-Arg mutated Ub genes as their sole source of Ub (Figure 6B). The profiles of Sna3p–Ub conjugates were identical in cells producing wild-type Ub or mutated Ub with substitutions at Lys6, 11, 27, 29 and 33. The effect of substituting Lys48 was not tested because it is lethal for the cells (36). In contrast, the pattern of Ub–Sna3p conjugates in cells producing UbK63R as sole source of Ub was similar to that observed in doa4Δ cells overexpressing UbK63R, with the number of Ub conjugates notably reduced. The presence of more than one Ub-conjugated band still remains to be understood, but it is possible that when Lys63-Ub chains cannot be formed, an alternative chain is synthesized, at least in this case. Whatever the case, the overall data clearly indicate that Sna3p is modified by Lys63-Ub chains.

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