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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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Expression of different marker proteins in MPR-deficient fibroblasts. Total cell extracts of MPR-positive or MPRnegative fibroblasts and MPR-negative fibroblasts reexpressing  either MPR were prepared as described in Materials and Methods.  Similar amounts of proteins (30 μg) were resolved on SDS-PAGE,  transferred onto nitrocellulose, and sequentially analyzed by Western blotting for their content in γ-adaptin (arrow), α-adaptin,  transferrin receptor (Tf. Rec.), β-COP (coatomer), and Lamp-1  (A). The γ-adaptin signal was quantitated and then normalized to  that of the α-adaptin (B) or transferrin receptor (C) as indicated  in Materials and Methods. MPR −/−, MPR-negative fibroblasts;  MPR +/+, control fibroblasts expressing the two MPRs; MOCK,  mock-transfected MPR-negative cells; CD-4, -1, and -2, MPRnegative fibroblasts reexpressing 1.5-, 3.5-, and 4.4-fold the endogenous level of CD-MPR, respectively; CI-3 and -4, MPR-negative fibroblasts reexpressing one- and fivefold the endogenous  level of CI-MPR, respectively. Values correspond to means ±  standard error of four independent experiments. When compared with mock-transfected MPR-negative fibroblasts, the sample populations were not found to be significantly different according to the t test.
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Figure 3: Expression of different marker proteins in MPR-deficient fibroblasts. Total cell extracts of MPR-positive or MPRnegative fibroblasts and MPR-negative fibroblasts reexpressing either MPR were prepared as described in Materials and Methods. Similar amounts of proteins (30 μg) were resolved on SDS-PAGE, transferred onto nitrocellulose, and sequentially analyzed by Western blotting for their content in γ-adaptin (arrow), α-adaptin, transferrin receptor (Tf. Rec.), β-COP (coatomer), and Lamp-1 (A). The γ-adaptin signal was quantitated and then normalized to that of the α-adaptin (B) or transferrin receptor (C) as indicated in Materials and Methods. MPR −/−, MPR-negative fibroblasts; MPR +/+, control fibroblasts expressing the two MPRs; MOCK, mock-transfected MPR-negative cells; CD-4, -1, and -2, MPRnegative fibroblasts reexpressing 1.5-, 3.5-, and 4.4-fold the endogenous level of CD-MPR, respectively; CI-3 and -4, MPR-negative fibroblasts reexpressing one- and fivefold the endogenous level of CI-MPR, respectively. Values correspond to means ± standard error of four independent experiments. When compared with mock-transfected MPR-negative fibroblasts, the sample populations were not found to be significantly different according to the t test.

Mentions: We first determined by quantitative Western blotting the level of expression of different markers in MPR-negative fibroblasts and in MPR-negative fibroblasts stably reexpressing physiological levels of either the cation-dependent (CD) MPR or the insulin-like growth factor II/CIMPR (Ludwig et al., 1994; Le Borgne et al., 1996; Mauxion et al., 1996). Fig. 3 shows the relative amounts of γ-adaptin (AP-1), α-adaptin (AP-2), lysosomal marker Lamp-1, or transferrin receptor, a membrane protein recycling between the plasma membrane and the endosomes, as well as that of β-COP, a subunit of the coatomer chosen as a marker of the early secretory pathway in the total cell lysates of the different clones examined. The expression level of each of these markers was nearly identical in all of the different cell types examined. In addition, the ratios of γ-adaptin/α-adaptin (Fig. 3 B), γ-adaptin/transferrin receptor (Fig. 3 C), or γ-adaptin/β-COP (not shown) were very similar in all of the different cell types examined. This indicates that the expression level of these different markers is, as expected, independent of the expression of the MPRs.


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

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

Expression of different marker proteins in MPR-deficient fibroblasts. Total cell extracts of MPR-positive or MPRnegative fibroblasts and MPR-negative fibroblasts reexpressing  either MPR were prepared as described in Materials and Methods.  Similar amounts of proteins (30 μg) were resolved on SDS-PAGE,  transferred onto nitrocellulose, and sequentially analyzed by Western blotting for their content in γ-adaptin (arrow), α-adaptin,  transferrin receptor (Tf. Rec.), β-COP (coatomer), and Lamp-1  (A). The γ-adaptin signal was quantitated and then normalized to  that of the α-adaptin (B) or transferrin receptor (C) as indicated  in Materials and Methods. MPR −/−, MPR-negative fibroblasts;  MPR +/+, control fibroblasts expressing the two MPRs; MOCK,  mock-transfected MPR-negative cells; CD-4, -1, and -2, MPRnegative fibroblasts reexpressing 1.5-, 3.5-, and 4.4-fold the endogenous level of CD-MPR, respectively; CI-3 and -4, MPR-negative fibroblasts reexpressing one- and fivefold the endogenous  level of CI-MPR, respectively. Values correspond to means ±  standard error of four independent experiments. When compared with mock-transfected MPR-negative fibroblasts, the sample populations were not found to be significantly different according to the t test.
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Related In: Results  -  Collection

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Figure 3: Expression of different marker proteins in MPR-deficient fibroblasts. Total cell extracts of MPR-positive or MPRnegative fibroblasts and MPR-negative fibroblasts reexpressing either MPR were prepared as described in Materials and Methods. Similar amounts of proteins (30 μg) were resolved on SDS-PAGE, transferred onto nitrocellulose, and sequentially analyzed by Western blotting for their content in γ-adaptin (arrow), α-adaptin, transferrin receptor (Tf. Rec.), β-COP (coatomer), and Lamp-1 (A). The γ-adaptin signal was quantitated and then normalized to that of the α-adaptin (B) or transferrin receptor (C) as indicated in Materials and Methods. MPR −/−, MPR-negative fibroblasts; MPR +/+, control fibroblasts expressing the two MPRs; MOCK, mock-transfected MPR-negative cells; CD-4, -1, and -2, MPRnegative fibroblasts reexpressing 1.5-, 3.5-, and 4.4-fold the endogenous level of CD-MPR, respectively; CI-3 and -4, MPR-negative fibroblasts reexpressing one- and fivefold the endogenous level of CI-MPR, respectively. Values correspond to means ± standard error of four independent experiments. When compared with mock-transfected MPR-negative fibroblasts, the sample populations were not found to be significantly different according to the t test.
Mentions: We first determined by quantitative Western blotting the level of expression of different markers in MPR-negative fibroblasts and in MPR-negative fibroblasts stably reexpressing physiological levels of either the cation-dependent (CD) MPR or the insulin-like growth factor II/CIMPR (Ludwig et al., 1994; Le Borgne et al., 1996; Mauxion et al., 1996). Fig. 3 shows the relative amounts of γ-adaptin (AP-1), α-adaptin (AP-2), lysosomal marker Lamp-1, or transferrin receptor, a membrane protein recycling between the plasma membrane and the endosomes, as well as that of β-COP, a subunit of the coatomer chosen as a marker of the early secretory pathway in the total cell lysates of the different clones examined. The expression level of each of these markers was nearly identical in all of the different cell types examined. In addition, the ratios of γ-adaptin/α-adaptin (Fig. 3 B), γ-adaptin/transferrin receptor (Fig. 3 C), or γ-adaptin/β-COP (not shown) were very similar in all of the different cell types examined. This indicates that the expression level of these different markers is, as expected, independent of the expression of the MPRs.

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