Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN - PubMed (original) (raw)
Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN
R Le Borgne et al. J Cell Biol. 1997.
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.
Figures
Figure 1
AP-1 immunostaining in MPR-positive and MPR-negative mouse fibroblasts. MPR-positive and MPR-negative fibroblasts were cocultured on coverslips, fixed, and processed for double immunofluorescence using an mAb against Lamp-1 taken as a lysosomal marker (A) and a polyclonal antibody against the hinge region of the mouse γ-adaptin (B) as indicated in Materials and Methods. The MPR-negative fibroblasts are easily identified by the large number of Lamp-1–positive structures; (stars) MPR-positive fibroblasts.
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.
Figure 5
Distribution of marker proteins on linear Ficoll/2H2O density gradients. Clathrin-coated vesicles were purified from mock-transfected MPR-negative fibroblasts (A) and MPR-negative fibroblasts reexpressing the CI-MPR (clone CI-4) (B) as described in Materials and Methods. Each fraction from the last linear density gradient was analyzed by Western blotting for its content in CI-MPR, γ-adaptin (arrow), α-adaptin, transferrin receptor (Tf Rec.), and clathrin light chains (Clathrin L.C.).
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.
Figure 4
Membrane-bound γ-adaptin and MPR expression. Microsomal membranes of MPR-deficient fibroblasts were prepared as described in Materials and Methods and analyzed by Western blotting for their content in γ-adaptin (arrow), α-adaptin, transferrin receptor, or β-COP (A). The amount of γ-adaptin bound to microsomal membranes was then quantitated and normalized to that of α-adaptin (B) or transferrin receptor (C). (MPR −/−, MPR-negative fibroblasts; MPR +/+, control fibroblasts expressing the two MPRs; MOCK, mock-transfected MPRnegative cells; CD-4, -1, and -2, MPR-negative 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 represent means ± standard error of four independent experiments. When MPR-positive fibroblasts or MPR-negative fibroblasts reexpressing either MPR were compared with the mock-transfected MPR-negative fibroblasts, the confidence limits of the sample populations were found to be >99% in every case based on the t test.
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.
Figure 7
AP-1–coated vesicles and MPR expression. Clathrincoated vesicles were isolated from the different MPR-negative fibroblasts, and their content in γ-adaptin, α-adaptin, and transferrin receptor was analyzed by quantitative Western blotting as shown in Fig. 4. The amount of γ-adaptin was then normalized to that of α-adaptin (A) or transferrin receptor (B). MPR −/−, MPR-negative fibroblasts; MPR +/+, control fibroblasts expressing the two MPRs; MOCK, mock-transfected MPR-negative fibroblasts; CD-4, -1, and -2, MPR-negative 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 the mean ± standard error of two (CD-1 and CI-3) and three (CD-4, CD-2, and CI-4) independent experiments. When MPRpositive fibroblasts or MPR-negative fibroblasts reexpressing either MPR were compared with the mock-transfected MPR-negative fibroblasts, the confidence limits of the sample populations were found to be >99% in every case based on the t test.
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.
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