Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of the polyclonal anti–γ-adaptin antibody. (A) Postnuclear supernatants (lane 1) or total membranes  (lane 2) were prepared from mouse fibroblasts. The samples were  fractionated by SDS-PAGE, transferred to nitrocellulose, and  analyzed by Western blotting using a polyclonal antibody against  a peptide corresponding to the trunk region (1573; right) or  against the hinge region of the mouse γ-adaptin (γ-H; left). (Arrow) γ-Adaptin; (arrowhead) a 120-kD contaminant. (B) γ-Adaptin  was immunoprecipitated from denaturated (left) or native (right)  HeLa cell lysates with either the polyclonal antibody (1573) or  with the mAb 100/3 as described in Materials and Methods. Both  the immunoprecipitated material (P) and a fraction (10%) of the  supernatant of the immunoprecipitations (S) were analyzed by  Western blotting with either the 1573 or the 100/3 antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139777&req=5

Figure 2: Characterization of the polyclonal anti–γ-adaptin antibody. (A) Postnuclear supernatants (lane 1) or total membranes (lane 2) were prepared from mouse fibroblasts. The samples were fractionated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting using a polyclonal antibody against a peptide corresponding to the trunk region (1573; right) or against the hinge region of the mouse γ-adaptin (γ-H; left). (Arrow) γ-Adaptin; (arrowhead) a 120-kD contaminant. (B) γ-Adaptin was immunoprecipitated from denaturated (left) or native (right) HeLa cell lysates with either the polyclonal antibody (1573) or with the mAb 100/3 as described in Materials and Methods. Both the immunoprecipitated material (P) and a fraction (10%) of the supernatant of the immunoprecipitations (S) were analyzed by Western blotting with either the 1573 or the 100/3 antibodies.

Mentions: Our previous in vitro studies on MPR-deficient mouse fibroblasts have strongly suggested that the MPRs are key components for the efficient translocation of cytosolic AP-1 onto membranes. These studies were based on an in vitro assay reconstituting the binding of cytosolic bovine AP-1 on membranes of permeabilized mouse fibroblasts. The newly bound AP-1 was detected with the 100/3 anti– γ-adaptin mAb, unable to recognize the endogenous mouse AP-1 (Le Borgne et al., 1993, 1996). When labeled with an antibody against the hinge region of γ-adaptin (Seaman et al., 1996), the mouse fibroblasts expressing the two MPRs exhibit the typical, perinuclear γ-adaptin staining (Fig. 1). In contrast, the fibroblasts devoid of the MPRs, easily identified by the large number of Lamp-1–positive structures as a result of the massive missorting of lysosomal enzymes (Ludwig et al., 1994), show a reduced γ-adaptin staining (Fig. 1). To quantitate by Western blotting the steady state distribution of γ-adaptin in these cells, we have used a polyclonal antibody against a peptide corresponding to a stretch of amino acids (R259–Q274) contained in the trunk domain of the mouse γ-adaptin, also conserved in the human γ-adaptin. Fig. 2 A shows that, in Western blot analysis, this anti–mouse γ-adaptin antibody reacts with two proteins of mouse fibroblasts. The first protein is the ∼100-kD γ-adaptin because it is also recognized by a polyclonal antibody directed against the hinge region of γ-adaptin, while the second, ∼120-kD protein, not recognized by the polyclonal antibody against the hinge region of the mouse γ-adaptin and absent from purified clathrin-coated vesicles (see Fig. 5), most likely represents a contaminant. Furthermore, Fig. 2 B shows that this anti-peptide antibody immunoprecipitates the γ-adaptin from a total, denatured Hela cell lysate as does the 100/3 mAb. However, this antibody does not immunoprecipitate the native protein.


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 the polyclonal anti–γ-adaptin antibody. (A) Postnuclear supernatants (lane 1) or total membranes  (lane 2) were prepared from mouse fibroblasts. The samples were  fractionated by SDS-PAGE, transferred to nitrocellulose, and  analyzed by Western blotting using a polyclonal antibody against  a peptide corresponding to the trunk region (1573; right) or  against the hinge region of the mouse γ-adaptin (γ-H; left). (Arrow) γ-Adaptin; (arrowhead) a 120-kD contaminant. (B) γ-Adaptin  was immunoprecipitated from denaturated (left) or native (right)  HeLa cell lysates with either the polyclonal antibody (1573) or  with the mAb 100/3 as described in Materials and Methods. Both  the immunoprecipitated material (P) and a fraction (10%) of the  supernatant of the immunoprecipitations (S) were analyzed by  Western blotting with either the 1573 or the 100/3 antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139777&req=5

Figure 2: Characterization of the polyclonal anti–γ-adaptin antibody. (A) Postnuclear supernatants (lane 1) or total membranes (lane 2) were prepared from mouse fibroblasts. The samples were fractionated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting using a polyclonal antibody against a peptide corresponding to the trunk region (1573; right) or against the hinge region of the mouse γ-adaptin (γ-H; left). (Arrow) γ-Adaptin; (arrowhead) a 120-kD contaminant. (B) γ-Adaptin was immunoprecipitated from denaturated (left) or native (right) HeLa cell lysates with either the polyclonal antibody (1573) or with the mAb 100/3 as described in Materials and Methods. Both the immunoprecipitated material (P) and a fraction (10%) of the supernatant of the immunoprecipitations (S) were analyzed by Western blotting with either the 1573 or the 100/3 antibodies.
Mentions: Our previous in vitro studies on MPR-deficient mouse fibroblasts have strongly suggested that the MPRs are key components for the efficient translocation of cytosolic AP-1 onto membranes. These studies were based on an in vitro assay reconstituting the binding of cytosolic bovine AP-1 on membranes of permeabilized mouse fibroblasts. The newly bound AP-1 was detected with the 100/3 anti– γ-adaptin mAb, unable to recognize the endogenous mouse AP-1 (Le Borgne et al., 1993, 1996). When labeled with an antibody against the hinge region of γ-adaptin (Seaman et al., 1996), the mouse fibroblasts expressing the two MPRs exhibit the typical, perinuclear γ-adaptin staining (Fig. 1). In contrast, the fibroblasts devoid of the MPRs, easily identified by the large number of Lamp-1–positive structures as a result of the massive missorting of lysosomal enzymes (Ludwig et al., 1994), show a reduced γ-adaptin staining (Fig. 1). To quantitate by Western blotting the steady state distribution of γ-adaptin in these cells, we have used a polyclonal antibody against a peptide corresponding to a stretch of amino acids (R259–Q274) contained in the trunk domain of the mouse γ-adaptin, also conserved in the human γ-adaptin. Fig. 2 A shows that, in Western blot analysis, this anti–mouse γ-adaptin antibody reacts with two proteins of mouse fibroblasts. The first protein is the ∼100-kD γ-adaptin because it is also recognized by a polyclonal antibody directed against the hinge region of γ-adaptin, while the second, ∼120-kD protein, not recognized by the polyclonal antibody against the hinge region of the mouse γ-adaptin and absent from purified clathrin-coated vesicles (see Fig. 5), most likely represents a contaminant. Furthermore, Fig. 2 B shows that this anti-peptide antibody immunoprecipitates the γ-adaptin from a total, denatured Hela cell lysate as does the 100/3 mAb. However, this antibody does not immunoprecipitate the native protein.

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