Association of Dynamin with the Golgi Apparatus
Association of a dynamin-like protein with the Golgi apparatus in mammalian cells
J Cell Biol 1996 May; 133(4): 761-75
Dynamins are a family of 100-kD GTPases comprised of at least three distinct gene products and multiple alternatively spliced variants. Homologies with the shibire gene product in Drosophila melanogaster and with Vps1p and Dnm1p in Saccharomyces cerevisiae suggest that dynamins play an important role in vesicular transport. Morphological studies have localized brain dynamin to coated pits and tubular invaginations at the plasma membrane, where it is believed to facilitate the formation of endocytic vesicles. Because similar membrane-budding events occur at the Golgi apparatus and multiple dynamin isoforms exist, we have studied the distribution of dynamins in mammalian cells.
To this end, we generated and characterized peptide-specific antibodies directed against conserved regions of the dynamin family. By immunoblot analysis, these antibodies reacted specifically with a 100-kD protein in fibroblasts that sedimented with membranes and microtubules in vitro in a manner similar to brain dynamin. By immunofluorescence microscopy, these antibodies strongly labeled the Golgi complex in cultured fibroblasts and melanocytes, as confirmed by double labeling with a Golgi-specific antibody. Furthermore, Western blot analysis showed significant enrichment of a 100-kD dynamin band in Golgi fractions isolated from the liver. To substantiate these findings, we use a specific antidynamin antibody to immunoisolate Golgi membranes from subcellular Golgi fractions, as determined by EM and immunoblot analysis.
This study provides the first morphological and biochemical evidence that a dynamin-like protein associates with the Golgi apparatus in mammalian cells, and suggests that dynamin-related proteins may have multiple cytoplasmic distributions. The potential contributions of dynamin to the secretory and endocytic pathways are discussed.
Multiple antibodies to dynamin label the Golgi apparatus in fibroblasts
Cultured human fibroblasts were processed for double-label immunofluorescence microscopy using three different affinity-purified antibodies to dynamin: MC12 (a), MC13 (b), MC63 (c). (a'-c') The Golgi-specific mAb 10E6. Note the strong co-localization of dynamin immunoreactivity (green) and the Golgi apparatus (red). Identical staining patterns are seen with two other anti-dynamin antibodies (MC14 and MC62) not shown. In addition to the Golgi stain, punctate vesicular structures are also seen. No immunofluorescence staining was detected when pre-immune sera or void volumes from the antibody affinity purification step were used (data not shown). (a''-c'') Corresponding phase contrast images. Bars, 20 um.
Dynamin is enriched in subcellular Golgi fractions as indicated by Western blot analysis
(A) Anti-dynamin antibody MC63 was used for Western blot analysis of subcellular fractions from liver representing various stages of Golgi purification. These fractions include a post-nuclear supernatant (S1), a total membrane pellet (TM), a crude membrane fraction (CM) obtained from the first discontinuous sucrose gradient centrifugation, and a mixed Golgi fraction (G) obtained after a second discontinuous sucrose gradient centrifugation. Highly enriched fractions containing Golgi vesicles (Gv) or intact Golgi stacks (Gs) were obtained upon further subfractionation of the mixed Golgi fraction (see Materials and Methods). Fractions were characterized using antibodies against three different Golgi marker proteins, alpha-mannosidase II (Mann II), beta-COP, and rab 6; the RER-associated protein, ribophorin II (Ribo II); and the polymeric IgA receptor (IgA rec).
A dynamin-immunoreactive band that is detected in the post-nuclear supernatant migrates with purified brain dynamin (D, 1 µg/lane). This dynamin-immunoreactive protein is de-enriched in the total membrane pellet, indicating that it does not preferentially associate with membranes. Upon enriching for Golgi membranes, as determined by the increased immunoreactivity for alpha-mannosidase II, beta-COP, rab 6, and the 105 kD form of the polymeric IgA receptor (IgA rec, lower band), there is a concomitant increase in dynamin immunoreactivity, with the greatest enrichment in the stacked Golgi fraction. This stacked Golgi fraction has little ribophorin II immunoreactivity, indicating that RER is not enriched. Also, note that there is no increase in the immunoreactivity for the 120 kD form of the polymeric IgA receptor (IgA rec 120, upper band), indicating that endocytic compartments, likewise, are not enriched in this stacked Golgi fraction.
Lines to the lower right of the figure indicate the positions, from top to bottom, of the 120, 116, and 105 kD forms of the polymeric IgA receptor, respectively. An equal amount of protein was loaded for each liver fraction (50 µg/lane). (B, C) Electron micrographs demonstrating numerous Golgi profiles in the isolated stacked Golgi fraction, including stacked Golgi elements (asterisks), and lipoprotein-containing vesicles (arrowheads). Coated buds and vesicles can also be seen (C, small arrows). Bars, 0.3 um.
Golgi membranes are immunoisolated using an antibody to dynamin
(A) Starting and immunoisolated fractions were analyzed by immunoblotting with an antibody to the Golgi protein alpha-mannosidase II. Golgi fractions (Golgi) were isolated from liver using two different established methods, and consisted of either a stacked Golgi fraction (Prep I), or a mixture of Golgi heavy and Golgi light membrane fractions (Prep II; see Materials and Methods). These fractions were incubated with beads that were coated with affinity-purified antibody MC63, and the material that bound (Beads) or did not bind (Unbnd) to the beads was collected.
Protein loads were equivalent to the starting Golgi fraction for each lane. There was a significant accumulation of the Golgi marker protein in the immunoisolated bead fractions, while very little remained in the unbound fractions. (B-D) Electron micrographs representative of the immunoisolated fraction obtained from the stacked Golgi fraction with antibody MC63-coated beads, as described above. Multiple Golgi profiles (asterisks), lipoprotein-containing vesicles (arrowheads), and fenestrated membranes associated with Golgi elements (C, D; open arrows) are clearly visible. (E) Electron micrograph representative of a control fraction obtained after incubating the stacked Golgi fraction with beads that were coated with an anti-rabbit IgG linker antibody alone. Dark granules are noncellular debris. Few, if any membranes are associated with the beads. Bars, 0.6 um (B, E) and 0.15 um (C, D).
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