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In Vitro Reconstitution of Microtubule Plus End-directed, GTPS-sensitive Motility ofGolgi Membranes

Aaron T. Fullerton,^* Mu-Yeh Bau,^* Patricia A. Conrad, and George S. Bloom^*

 ^*Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and  Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5'-adenylylimidodiphosphate, and 100 µM but not 20 µM vanadate. Motility was also blocked by GTPS or AlF₄ but was insensitive to AlCl₃, NaF, staurosporin, or okadaic acid. The targets for GTPS and AlF₄ were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.

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   Corresponding author. E-mail address: bloom{at}utsw.swmed.edu.
   Online version of this article contains video material for Figures 3 and 9. Online version available at www.molbiolcell.org.

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