Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus
Shin-ichiro Yoshimura1,2,
Nobuhiro Nakamura2,*,
Francis A. Barr3,
Yoshio Misumi4,
Yukio Ikehara4,
Hiroshi Ohno2,
Masao Sakaguchi1 and
Katsuyoshi Mihara1
1 Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
2 Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan
3 Department of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, Martinsried, D-82152, Germany
4 Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan
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Fig. 1. Newly synthesized GM130 and GRASP65 are quickly localized to the membrane. NRK cells stably expressing NAGFP were labeled with [35S]-methionine/cysteine for 5 minutes and chased for 0 minutes, 3 minutes, 7 minutes, 15 minutes and 30 minutes. Cells were homogenized and post-nuclear supernatant (PNS) was recovered. PNS (S) was further separated into total membrane (M) and cytosol (C) fractions. (A) GM130, GRASP65, calnexin and NAGFP were immunoprecipitated from each fraction and analyzed by SDS-PAGE and autoradiography. The immature form (im) and the mature form (m) of NAGFP are indicated. An asterisk indicates a non-specific precipitate. The picture shown is a representative of two experiments with similar results. (B) The percentage of GM130 (closed squares), GRASP65 (closed triangles) NAGFP (open circles) and calnexin (open diamonds) recovered in each membrane was plotted for the chase time. The plots are the average of two experiments and vertical bars indicate the ranges. (C) PNS was subjected to flotation as described in Materials and Methods and separated into cytosolic (C) and membrane (M) fractions. GM130 and GRASP65 were immunoprecipitated from each fraction and analyzed by SDS-PAGE and autoradiography. The picture shown is a representative of three experiments with similar results.
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Fig. 2. Newly synthesized GM130 and GRASP65 are localized to Golgi membranes. (A) NRK cells stably expressing NAGFP were labeled with [35S]-methionine/cysteine for 5 minutes and homogenized immediately (upper gallery) or after 30 minutes chase (lower gallery). Post-nuclear supernatant was obtained and analyzed by Nycodenz density gradient as described in Materials and Methods. To the left of the panels (lane 1) is the top (lightest) fraction and the right (lane 10) is the bottom (heaviest) fraction. GM130, GRASP65, calnexin and NAGFP were immunoprecipitated from each fraction and precipitated materials were analyzed by SDS-PAGE and autoradiography (indicated by arrows). Asterisks indicate nonspecific precipitates. The picture shown is a representative of three experiments with similar results. (B) Bands in (A) were quantified by densitometry and percentages of the total sum of the precipitated material in all fractions were calculated and plotted for each fraction.
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Fig. 3. Localization of newly synthesized GM130 and GRASP65 in the presence of a dominant negative form of Sar1p. (A) Sar1p H79G mutant (mSar1p; top panels) or buffer (mock; bottom panels) were first microinjected into the cytoplasm of NRK cells with cascade-blue-conjugated BSA as an injection marker. After 15 minutes incubation, plasmids encoding NAGFP and FLAG-GM130 were subsequently microinjected into the nuclei and the cells were further treated as described in Materials and Methods. Two-dimensional projections of triple range confocal microscope images are shown: NAGFP (GFP fluorescence; right panels), FLAG-GM130 (Cy3 staining; middle panels) and giantin (Cy5 staining; left panels). Asterisks indicate the microinjected cells. (B) As (A) except that GRAP65-HA was expressed in the middle panels. (C) mSar1p was microinjected into the cytoplasm of NRK cells with cascade-blue-conjugated BSA as an injection marker. After 100 minutes incubation, cells were fixed and subjected to the immunofluorescence staining. Top panels: giantin (left) and GM130 (right) were double stained. Bottom panels: mannosidase II (left) and GM130 (right) were double stained.
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Fig. 4. GM130 and GRASP65 are directly targeted to Golgi apparatus in vitro. (A) [35S]-labeled GM130 were synthesized in vitro using reticulocyte lysate and incubated with increasing amount of purified Golgi, mitochondrial or microsomal membranes as indicated. Membranes were recovered by centrifugation, the bound material was analyzed by SDS-PAGE and autoradiography. The picture shown is a representative of three experiments with similar results (top gallery). Amounts of membrane-bound GM130 and GRASP65 were quantified as described in Materials and Methods and plotted against the amounts of final membrane concentrations. The results shown were the average of three experiments and the vertical bars indicate standard deviations (bottom). (B) Experiments were carried out for GRASP65 as described in (A).
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Fig. 5. Mutant GM130 and GRASP65 do not bind to Golgi membranes in vitro. (A) Wild-type (Wt) or1 mutant forms ( C983 and M984A) of GM130 were synthesized in vitro and used for Golgi membrane binding assay as described in Fig. 4 (top). Amounts of membrane-bound GM130 were quantified as described in Materials and Methods (bottom). (B) Same as (A) except that wild-type (Wt) or mutant (G2A and G196A) forms of GRASP65 were used for the experiments.
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Fig. 6. Complex formation of in vitro membrane-bound GM130 and GRASP65 with pre-existing proteins. In vitro synthesized GM130 (A) and GRASP65 (B) were bound to Golgi membranes and the membranes were recovered as described in Fig. 4. The membranes were lysed and analyzed directly (T) or subjected to immunoprecipitation by anti-GM130 (GM, I), anti-GRASP65 (GR, I) antibodies. Control precipitation was performed with a pre-immune serum for each antibody (P). Bound materials were analyzed by SDS-PAGE followed by autoradiography.
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© The Company of Biologists Ltd 2001
