IN-VITRO RECONSTITUTION OF MICROTUBULE PLUS END-DIRECTED, GTP-GAMMA-S-SENSITIVE MOTILITY OF GOLGI MEMBRANES

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.