Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.
Cytoplasmic dynein is a microtubule minus-end-directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661-675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.
Fath
K R
KR
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Trimbur
G M
GM
Burgess
D R
DR
D000818:Animals; D017487:Ankyrins / Q000302:isolation & purification; D016764:Cell Polarity / Q000502:physiology*; D002474:Cell-Free System; D002645:Chickens; D020756:Coatomer Protein; D003600:Cytosol / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D004847:Epithelial Cells; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D007413:Intestinal Mucosa; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D008870:Microtubules / Q000502:physiology; D009068:Movement; D009218:Myosins / Q000302:isolation & purification; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D017487:Ankyrins; D020756:Coatomer Protein; D008869:Microtubule-Associated Proteins; D013049:Spectrin; D017830:Octoxynol; D009218:Myosins; D004398:Dyneins