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Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN.

Le Borgne R, Hoflack B - J. Cell Biol. (1997)

Bottom Line: Biol.Chem. 271:2162-2170).Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.

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Mutations in the CD-MPR cytoplasmic domain and formation of AP-1–coated vesicles. Microsomal membranes and clathrin-coated vesicles were prepared from MPR-negative fibroblasts  reexpressing either the wild-type CD-MPR (squares) or a CD-MPR  mutated on the casein kinase II phosphorylation site present in  its cytoplasmic domain (circles). The amounts of γ-adaptin bound  to membranes (A) and associated with AP-1–coated vesicles (B)  were determined by quantitative Western blotting and normalized to the amount of α-adaptin and transferrin receptor. The indicated values represent means ± SEM of three independent experiments. The 100% value corresponds to the amount of γ-adaptin  bound to microsomal membranes (A) or associated with clathrincoated vesicles (B) of MPR-negative fibroblasts used as controls.
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Figure 8: Mutations in the CD-MPR cytoplasmic domain and formation of AP-1–coated vesicles. Microsomal membranes and clathrin-coated vesicles were prepared from MPR-negative fibroblasts reexpressing either the wild-type CD-MPR (squares) or a CD-MPR mutated on the casein kinase II phosphorylation site present in its cytoplasmic domain (circles). The amounts of γ-adaptin bound to membranes (A) and associated with AP-1–coated vesicles (B) were determined by quantitative Western blotting and normalized to the amount of α-adaptin and transferrin receptor. The indicated values represent means ± SEM of three independent experiments. The 100% value corresponds to the amount of γ-adaptin bound to microsomal membranes (A) or associated with clathrincoated vesicles (B) of MPR-negative fibroblasts used as controls.

Mentions: Our previous in vitro analysis based on the reexpression of CD-MPR mutants in MPR-negative fibroblasts has indicated that the high affinity binding of AP-1 to membranes (Kd ∼40 nM) relies on the presence of specific determinants in the CD-MPR cytoplasmic domain (Mauxion et al., 1996). In particular, we observed that mutations introduced in the casein kinase II phosphorylation site present in its carboxyl-terminal domain (mutant A565859 according to Mauxion et al., 1996) significantly reduce the affinity of AP-1 for membranes (Kd ∼120 nM) without affecting the number of AP-1 binding sites. This mutant is also drastically, but not completely, impaired in proper transport of lysosomal enzymes in vivo (35% of sorting efficiency). Therefore, these stable clones gave us the opportunity to investigate the relationship between the affinity of AP-1 for its target membrane in vitro and its efficiency in producing TGN-derived vesicles in vivo. Toward this goal, a similar type of analysis, as described above, was performed on these MPR-negative fibroblasts reexpressing a CD-MPR mutated on the casein kinase II phosphorylation site. In these stable clones, the expression level of the different marker proteins (α-adaptin, γ-adaptin, β-COP, and transferrin receptor) was very similar to that of MPR-positive or MPR-negative fibroblasts (not shown). Fig. 8 shows that the amount of γ-adaptin recovered at steady state bound to membranes or recovered in clathrin-coated vesicle fractions was far less abundant in MPR-negative fibroblasts reexpressing this CD-MPR mutant than in those reexpressing similar levels of wild-type CD-MPR. However, the amount of γ-adaptin bound to membranes or recovered associated with purified clathrin-coated vesicles was slightly higher in these cells than in MPR-negative fibroblasts. These data show that this CD-MPR mutant is unable to efficiently recruit AP-1 on membranes and to produce TGN-derived clathrin-coated vesicles. Thus, this CD-MPR mutant must be largely excluded from the AP-1–dependent pathway toward the endosomes. These results further highlight the importance of the casein kinase II phosphorylation site in the AP-1–dependent sorting of the CD-MPR and suggest that only high affinity interactions of AP-1 with its target membrane determine the production of AP-1 and clathrin-coated vesicles in vivo.


Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN.

Le Borgne R, Hoflack B - J. Cell Biol. (1997)

Mutations in the CD-MPR cytoplasmic domain and formation of AP-1–coated vesicles. Microsomal membranes and clathrin-coated vesicles were prepared from MPR-negative fibroblasts  reexpressing either the wild-type CD-MPR (squares) or a CD-MPR  mutated on the casein kinase II phosphorylation site present in  its cytoplasmic domain (circles). The amounts of γ-adaptin bound  to membranes (A) and associated with AP-1–coated vesicles (B)  were determined by quantitative Western blotting and normalized to the amount of α-adaptin and transferrin receptor. The indicated values represent means ± SEM of three independent experiments. The 100% value corresponds to the amount of γ-adaptin  bound to microsomal membranes (A) or associated with clathrincoated vesicles (B) of MPR-negative fibroblasts used as controls.
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Related In: Results  -  Collection

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Figure 8: Mutations in the CD-MPR cytoplasmic domain and formation of AP-1–coated vesicles. Microsomal membranes and clathrin-coated vesicles were prepared from MPR-negative fibroblasts reexpressing either the wild-type CD-MPR (squares) or a CD-MPR mutated on the casein kinase II phosphorylation site present in its cytoplasmic domain (circles). The amounts of γ-adaptin bound to membranes (A) and associated with AP-1–coated vesicles (B) were determined by quantitative Western blotting and normalized to the amount of α-adaptin and transferrin receptor. The indicated values represent means ± SEM of three independent experiments. The 100% value corresponds to the amount of γ-adaptin bound to microsomal membranes (A) or associated with clathrincoated vesicles (B) of MPR-negative fibroblasts used as controls.
Mentions: Our previous in vitro analysis based on the reexpression of CD-MPR mutants in MPR-negative fibroblasts has indicated that the high affinity binding of AP-1 to membranes (Kd ∼40 nM) relies on the presence of specific determinants in the CD-MPR cytoplasmic domain (Mauxion et al., 1996). In particular, we observed that mutations introduced in the casein kinase II phosphorylation site present in its carboxyl-terminal domain (mutant A565859 according to Mauxion et al., 1996) significantly reduce the affinity of AP-1 for membranes (Kd ∼120 nM) without affecting the number of AP-1 binding sites. This mutant is also drastically, but not completely, impaired in proper transport of lysosomal enzymes in vivo (35% of sorting efficiency). Therefore, these stable clones gave us the opportunity to investigate the relationship between the affinity of AP-1 for its target membrane in vitro and its efficiency in producing TGN-derived vesicles in vivo. Toward this goal, a similar type of analysis, as described above, was performed on these MPR-negative fibroblasts reexpressing a CD-MPR mutated on the casein kinase II phosphorylation site. In these stable clones, the expression level of the different marker proteins (α-adaptin, γ-adaptin, β-COP, and transferrin receptor) was very similar to that of MPR-positive or MPR-negative fibroblasts (not shown). Fig. 8 shows that the amount of γ-adaptin recovered at steady state bound to membranes or recovered in clathrin-coated vesicle fractions was far less abundant in MPR-negative fibroblasts reexpressing this CD-MPR mutant than in those reexpressing similar levels of wild-type CD-MPR. However, the amount of γ-adaptin bound to membranes or recovered associated with purified clathrin-coated vesicles was slightly higher in these cells than in MPR-negative fibroblasts. These data show that this CD-MPR mutant is unable to efficiently recruit AP-1 on membranes and to produce TGN-derived clathrin-coated vesicles. Thus, this CD-MPR mutant must be largely excluded from the AP-1–dependent pathway toward the endosomes. These results further highlight the importance of the casein kinase II phosphorylation site in the AP-1–dependent sorting of the CD-MPR and suggest that only high affinity interactions of AP-1 with its target membrane determine the production of AP-1 and clathrin-coated vesicles in vivo.

Bottom Line: Biol.Chem. 271:2162-2170).Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.

Show MeSH
Related in: MedlinePlus