Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin.
The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.
Cao
H
H
Department of Biochemistry and Molecular Biology & Center for Basic Research in Digestive Diseases, Mayo Clinic and Graduate School, Rochester, Minnesota 55905, USA.
Thompson
H M
HM
Krueger
E W
EW
McNiven
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D034281:Dynamins