MOLECULAR MOTORS AND A SPECTRIN MATRIX ASSOCIATE WITH GOLGI MEMBRANESIN-VITRO

Cytoplasmic dynein is a microtubule minus-end-directed motor that is t hought to power the transport of vesicles from the TGN to the apical c ortex in polarized epithelial cells. Trans-Golgi enriched membranes, w hich were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isola ted Golgi stacks lack dynein but-contain myosin-I (Fath, K.R., G.M. Tr imbur, 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 a bsence 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 st acks after budding. Budded membranes move exclusively towards microtub ule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resi sts extraction by ice-cold Triton X-100. In the presence of cytosol, t hese membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are clos ely associated with an amorphous matrix composed in part of spectrin a nd ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to ext racted membranes independently of spectrin and ankyrin.