At. Fullerton et al., IN-VITRO RECONSTITUTION OF MICROTUBULE PLUS END-DIRECTED, GTP-GAMMA-S-SENSITIVE MOTILITY OF GOLGI MEMBRANES, Molecular biology of the cell, 9(10), 1998, pp. 2699-2714
Purified Golgi membranes were mixed with cytosol and microtubules (MTs
) and observed by video enhanced light microscopy. Initially, the memb
ranes appeared as vesicles that moved along MTs. As time progressed, v
esicles formed aggregates from which membrane tubules emerged, travele
d along MTs, and eventually generated extensive reticular networks. Me
mbrane motility required ATP, occurred mainly toward MT plus ends, and
was inhibited almost completely by the H1 monoclonal antibody to kine
sin heavy chain, 5'-adenylylimidodiphosphate, and 100 mu M but not 20
mu M vanadate. Motility was also blocked by GTP gamma S or AlF4- but w
as insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targe
ts for GTP gamma S and AlF4- were evidently of cytosolic origin, did n
ot include kinesin or MTs, and were insensitive to several probes for
trimeric G proteins. Transport of Golgi membranes along MTs mediated b
y a kinesin has thus been reconstituted in vitro. The motility is regu
lated by one or more cytosolic GTPases but not by protein kinases or p
hosphatases that are inhibited by staurosporin or okadaic acid, respec
tively. The pertinent GTPases are likely to be small G proteins or pos
sibly dynamin. The in vitro motility may correspond to Golgi-to-ER or
Golgi-to-cell surface transport in vivo.