Role of Dynamin in the Formation of Transport Vesicles from the Trans-Golgi Network
Science; 23 January 1998; 279: 573-577
Steven M. Jones, Kathryn E. Howell, John R. Henley, Hong Cao, Mark A. McNiven
Dynamin guanosine triphosphatases support the scission of clathrin-coated vesicles from the plasmalemma during endocytosis. By fluorescence microscopy of cultured rat hepatocytes, a green fluorescent protein-dynamin II fusion protein localized with clathrin-coated vesicles at the Golgi complex. A cell-free assay was utilized to demonstrate the role of dynamin in vesicle formation at the trans-Golgi. Addition of peptide-specific anti-dynamin antibodies to the assay mixture inhibited both constitutive exocytic and clathrin-coated vesicle formation. Immunodepletion of dynamin proteins also inhibited vesicle formation, and budding efficiency was restored upon readdition of purified dynamin. These data suggest that dynamin participates in the formation of distinct transport vesicles from the trans-Golgi network.
Figure 3 Antibodies to dynamin inhibit vesicle formation from the Golgi complex.
(A) Dynamin binding to a highly enriched Golgi fraction. The SGF1 was incubated with a cytosolic fraction in the ab-sence of ATP (lane 1); in the presence of ATP and an ATP-regenerating system (lane 2); or in the presence of ATP, an ATP-regenerating system, and 10 mM GTP-g-S (lane 3) at 37°C for 15 min. The reaction mixture was centrifuged through a sucrose cushion and the Golgi pellet was immunoblotted and analyzed for the presence of dy-namin. The dynamin-immunoreactive band at 100 kD is shown in the upper panel, and quantitation of the amount of dynamin bound (PhosphorIm-ager units) is shown in the lower panel. Note the increase in the association of dynamin with the Golgi membranes in the presence of either ATP or GTP-g-S. Each assay was carried out in triplicate and the standard error is plotted. (B) Antibodies to dynamin inhibit formation of both the pIgA-R containing exocytic and clathrin-coated vesicles. ( Top) Domains of Dyn2 are diagrammed and include three GTP-binding consensus sequence elements in the NH 2 -terminus, a pleckstrin homology (PH) domain in the COOH-terminal region, and a proline-rich domain at the COOH-terminus. The regions used to generate the polyclonal MC60, MC63, and Dyn2-specific antibodies are noted. The MC60 and MC63 epitopes are present in all dynamin isoforms, whereas the Dyn2-specific epitope is unique to Dyn2. (Middle and bottom) Cell-free assays of vesicle budding from the TGN were carried out in the presence of increasing amounts (0 to 16 mg) of affinity-purified antibodies. The budding efficiencies of the pIgA-R containing vesicles (middle) and clathrin-coated vesicles (bottom) are plotted against the antibody concentration. Control antibodies to the pIgA-R (not shown) and kinesin heavy chain (MC44) have no effect on vesicle budding, whereas the MC63 and Dyn2-specific antibodies were strongly inhibitory. Antibody MC60 induced a more modest inhibition. Antibodies were preincubated with the cytosolic fraction for 30 min on ice before they were added to the cell-free assay mixture. The antibody used in each reaction is listed above the corresponding graph: MC44 (against kinesin), MC60, MC63, and DYN2.
Figure 4 Vesicle formation from the TGN is dependent on dynamin and cytosolic factors.
(A) Immunodepletion of dynamin from rat liver cytosol using immunoaffinity columns. A rat liver cytosolic fraction was passed over two successive antibody columns and the nonbound fractions were collected and concentrated. Fractions of the starting (SM) and immunodepleted (DPL) cytosolic fraction were resolved by SDS-PAGE and stained with Coomassie blue (top) or transferred to nitrocellulose filters and blotted with the Pan-dynamin antibody MC63 (bottom). (B) Generation of an enriched dynamin fraction from rat brain for use in restoration experiments. A rat brain dynamin-enriched fraction (elute) was obtained by ion-exchange column chromatography as described (9). Proteins were resolved by SDS-PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose filters for immunoblot analysis (bottom). Other fractions shown are a high-speed supernatant (HSS) and the void volumes collected from DEAE (DE) and phosphocellulose (PC) anion-exchange columns. (C and D) Dynamin-dependent vesicle budding from the TGN by a reconstituted cell-free assay. Cell-free assays to measure budding of pIgA-R containing (C) and clathrin-coated (D) vesicles were carried out under the following conditions: in the absence of ATP and cytosol (lane 1); in the presence of ATP and cytosol (lane 2); with a dynamin-depleted cytosolic fraction (lane 3); with a dynamin-depleted cytosolic fraction plus increasing concentrations of a dynamin-enriched fraction (lanes 4 to 7); and with the dynamin-enriched fraction alone (lane 8). Lanes 4 to 7 contain 5, 10, 25, and 50 mg of dynamin-enriched fraction, respectively. Whereas no vesicle budding occurred with dynamin-depleted cytosol, formation of both pIgA-R containing (C) and clathrin-coated (D) vesicles was restored to near control levels when the dynamin fraction was added back to the reaction mixture. Importantly, the dynamin preparation alone did not support vesicle budding. Each assay was carried out in triplicate and the standard error is plotted. Immunoblot analysis of a representative experiment showing the 116-kD form of the pIgA-R (C) and the 180-kD clathrin heavy chain (D) is shown above each bar graph.
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