Association of a dynamin-like protein with the Golgi apparatus in mammalian cells.
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.
Henley
J R
JR
Department of Molecular Neuroscience, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
McNiven
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D029721:Drosophila Proteins; D034281:Dynamins; D005347:Fibroblasts / Q000166:cytology; D020558:GTP Phosphohydrolases / Q000276:immunology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008322:Mammals; D008544:Melanocytes / Q000166:cytology; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D029721:Drosophila Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D034281:Dynamins; C439628:shi protein, Drosophila