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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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Characterization of  clathrin-coated vesicles isolated from MPR-deficient fibroblasts. The material contained in fractions 8 to 10 of  the density gradients shown  in Fig. 5 B were concentrated  by centrifugation and analyzed. (A) Protein profile of  the vesicles isolated from  mouse fibroblasts after SDSPAGE and silver staining  (left). Clathrin-coated vesicles from bovine brain were  used for comparison (right).  (B) Clathrin-coated vesicles  purified from mouse fibroblasts observed by negative  staining. Bar, 100 nm.
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Figure 6: Characterization of clathrin-coated vesicles isolated from MPR-deficient fibroblasts. The material contained in fractions 8 to 10 of the density gradients shown in Fig. 5 B were concentrated by centrifugation and analyzed. (A) Protein profile of the vesicles isolated from mouse fibroblasts after SDSPAGE and silver staining (left). Clathrin-coated vesicles from bovine brain were used for comparison (right). (B) Clathrin-coated vesicles purified from mouse fibroblasts observed by negative staining. Bar, 100 nm.

Mentions: The membrane-bound γ-adaptin represents AP-1 bound to the donor compartment as well as AP-1 present in transport vesicles. Thus, clathrin-coated vesicles were prepared from the different MPR-deficient fibroblasts using the fractionation protocol described previously by Woodman and Warren (1991). Fig. 5 shows a typical example of the distribution of several marker proteins throughout the density gradient of the last purification step after fractionation of MPR-deficient fibroblasts. Typically, the dense fractions contained the clathrin light chain, the γ- and α-adaptins, as well as transmembrane proteins like the transferrin receptor and the CI-MPR, as determined by Western blotting. Very low amounts of β-COP were occasionally detected in the lighter fractions of this gradient (not shown). When analyzed by SDS-PAGE followed by protein staining, the dense fractions exhibited the typical protein profile of purified clathrin-coated vesicles (Fig. 6 A). Beside a few contaminants, the clathrin heavy and light chains as well as the different subunits of the APs were easily detected. At the morphological level, these fractions contained only spherical structures of ∼50–100 nm in diameter with a coat lattice reminiscent of clathrin-coated vesicles (Fig. 6 B).


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

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

Characterization of  clathrin-coated vesicles isolated from MPR-deficient fibroblasts. The material contained in fractions 8 to 10 of  the density gradients shown  in Fig. 5 B were concentrated  by centrifugation and analyzed. (A) Protein profile of  the vesicles isolated from  mouse fibroblasts after SDSPAGE and silver staining  (left). Clathrin-coated vesicles from bovine brain were  used for comparison (right).  (B) Clathrin-coated vesicles  purified from mouse fibroblasts observed by negative  staining. Bar, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139777&req=5

Figure 6: Characterization of clathrin-coated vesicles isolated from MPR-deficient fibroblasts. The material contained in fractions 8 to 10 of the density gradients shown in Fig. 5 B were concentrated by centrifugation and analyzed. (A) Protein profile of the vesicles isolated from mouse fibroblasts after SDSPAGE and silver staining (left). Clathrin-coated vesicles from bovine brain were used for comparison (right). (B) Clathrin-coated vesicles purified from mouse fibroblasts observed by negative staining. Bar, 100 nm.
Mentions: The membrane-bound γ-adaptin represents AP-1 bound to the donor compartment as well as AP-1 present in transport vesicles. Thus, clathrin-coated vesicles were prepared from the different MPR-deficient fibroblasts using the fractionation protocol described previously by Woodman and Warren (1991). Fig. 5 shows a typical example of the distribution of several marker proteins throughout the density gradient of the last purification step after fractionation of MPR-deficient fibroblasts. Typically, the dense fractions contained the clathrin light chain, the γ- and α-adaptins, as well as transmembrane proteins like the transferrin receptor and the CI-MPR, as determined by Western blotting. Very low amounts of β-COP were occasionally detected in the lighter fractions of this gradient (not shown). When analyzed by SDS-PAGE followed by protein staining, the dense fractions exhibited the typical protein profile of purified clathrin-coated vesicles (Fig. 6 A). Beside a few contaminants, the clathrin heavy and light chains as well as the different subunits of the APs were easily detected. At the morphological level, these fractions contained only spherical structures of ∼50–100 nm in diameter with a coat lattice reminiscent of clathrin-coated vesicles (Fig. 6 B).

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