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Volumn 279, Issue 5350, 1998, Pages 573-577

Role of dynamin in the formation of transport vesicles from the trans- Golgi network

Author keywords

[No Author keywords available]

Indexed keywords

CLATHRIN; DYNAMIN; GUANOSINE TRIPHOSPHATASE;

EID: 0032559342     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5350.573     Document Type: Article
Times cited : (282)

References (41)
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    • 6 cells per milliliter) with purified Dyn2 DNA (50 μg of DNA) (cuvette gap, 0.4 cm; voltage, 0.3 kV; capacitance, 250 μF) using a Bio-Rad instrument (Hercules, CA). After 24 hours, selection was initiated by addition of G418 (400 μg/ml) to the cell culture medium. A stable clone 9 cell line overexpressing GFP-Dyn2 was achieved within a month.
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    • Clone 9 cells were maintained at 37°C in Ham's F-12K medium supplemented with 10% fetal bovine serum. Cells were grown on cover glasses for 1 to 3 days before microscopy.
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    • For immunolocalization, cells were fixed in aldehyde and then labeled as described (9) and mounted in ProLong antifade reagent (Molecular Probes, Eugene, OR). Alternatively, live cells were viewed directly. Either an epifluorescence microscope (Axiovert 35, Carl Zeiss) equipped with a 100-W mercury arc (attenuated up to 90%) and a cooled charged coupled device (CCD) camera (SenSys, Photometrics, Tucson, AR) or a confocal laser scanning microscope (LSM-410, Carl Zeiss) was used for fluorescence microscopy.
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    • note
    • The location of each peptide used as antigen within the dynamin molecule is shown in Fig. 3B. The Pan-dynamin MC63 antibody has been shown to specifically recognize a 100-kD dynamin band in rat liver fractions by immunoblotting and immunoprecipitation (9). The Pan-dynamin MC60 and Dyn2-specific antibodies also have been characterized (27). The antibodies added to cell-free assays were affinity purified and concentrated (∼3 mg/ml); then they were tested by immunoblot analysis to confirm retention of activity (9). Antiserum against clathrin was produced from the hybridoma X22 (ATCC). The antibodies against the cytoplasmic and luminal domains of the plgA-R have been described (19). The rabbit polyclonal antibody to TGN38 was to a peptide representing the COOH-terminal cytoplasmic portion of the protein.
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    • 125I-labeled protein A (NEN, Boston, MA), and exposed to film for autoradiography. Immunoblots were quantitated with a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA).
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    • note
    • The cell-free assay of budding from immobilized stacked Golgi fractions was carried out as described (24). Each assay mixture contained a 2.5-mg magnetic core and shell beads with ∼50 μ9 of the stacked Golgi fraction immobilized. The immobilized fraction has been characterized (24). For the budding reaction, the immobilized fraction was incubated in 2.5 ml containing cytosol at 0.70 mg/ml, 25 mM Hepes (pH 6.7), 25 mM KCI, 1.5 mM magnesium acetate, 1.0 mM ATP, an ATP regenerating system (8.0 mM creatine phosphate, 0.043 mg of creatine phosphokinase per milliliter), and 5 mg of bovine serum albumin (BSA) per milliliter (final concentrations). For cell-free assays in which antibodies were tested, increasing concentrations of antibody were incubated with the cytosol for 30 min on ice before addition to the cell-free assay. After 10 min at 37°C the Golgi fraction remaining on the beads was retrieved, and the budded vesicles remained in the supernatant. The budded fraction was pelleted through a 0.25 M sucrose cushion (100,000g for 1 hour) to deplete the BSA (5 mg/ml) and large amounts of cytosolic proteins. The pellet was resuspended in gel sample buffer and resolved by SDS-PAGE. Budding efficiency was reported as the percentage of the total mature sialylated plgA-R (116 kD) present in the budded fraction (100% represents the amount present in the immobilized SGF before budding). The plgA-R distribution was determined by quantitative immunoblotting of the fractions from the cell-free assay. Because the plgA-R is a plasma membrane receptor synthesized in relatively high amounts in rat liver (28), it defines a specific population of constitutive exocytic vesicles (24). The amount of clathrin-coated vesicle formation was assessed by determining the amount of clathrin heavy chain in the total budded vesicle fraction by quantitative immunoblotting with monoclonal antibody TD.1 (ATCC). Percentage budding was calculated as the amount of clathrin heavy chain in the pelleted total budded fraction compared with that found in control budding reactions (100%). The amount of clathrin-coated vesicle budding in the absence of ATP and cytosol was 3%.
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    • For depletion of dynamin proteins from rat liver cytosol, 2 ml of rat liver cytosol (16 mg/ml), prepared by the methods of Palade and coworkers (28), was passed repeatedly over an MC63 Pan-dynamin antibody column at 4°C. The cytosolic void volume next was passed repeatedly over a Dyn2-specific antibody column at 4°C. The void volume was concentrated, separated by SDS-PAGE, and immunoblotted with dynamin antibodies to confirm a complete depletion of dynamin proteins from the cytosol. The dynamin antibody columns were prepared by immobilizing 9.3 mg and 4.9 mg of affinity-purified MC63 or Dyn2-specific antibodies, respectively, per 1.5-ml column matrix. All antibodies were immobilized by using an Immunopure protein A IgG orientation kit (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions.
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    • note
    • A dynamin-enriched fraction was isolated from freshly obtained rat brains according to established methods (9, 29). Briefly, a rat brain homogenate was passed through a 10-ml DEAE anion-exchange column and then added to a 5-ml phosphocellulose column. After substantial rinsing in 100 mM NaCI buffer, dynamin proteins were eluted from the column with 250 mM NaCl, and then the fractions were pooled, concentrated, dialyzed, and frozen in liquid nitrogen.
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    • note
    • At UCHSC the use of the Hepatobiliary Center, Cell Biology Core (NIH, P30 DK-34914), and the Cancer Center Monoclonal Antibody Facility (P30 CA-46934) is acknowledged. We especially thank J. Ugelstad and R. Schmid, SINTEF, University of Trondheim, Norway, for the shell and core magnetic beads used in the cell-tree assay. Thanks also goes to F. Garcia for her help with the GFP expression studies, to E. Krueger for his work on the GFP imaging, and to B. Oswald for help with preparation of antibodies and depleted cytosol.


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