Mackay, Deborah J. G.; (1993) Inhibition of intra-Golgi transport in mitotic extracts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Many stages of vesicle-mediated exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. The fact that transport between the ER and the Golgi is inhibited in mitosis made it technically very difficult to monitor mitotic intra-Golgi protein transport in vivo. Therefore, a cell-free assay was supplemented with heterologous cytosols to study transport inhibition in vitro. A high-speed supernatant ('cytosol') with high histone kinase activity was prepared from mitotic cells and markedly inhibited intra-Golgi transport. The inhibition was mimicked by treatment of interphase cytosol with the p34cdc-2-associated protein cyclin A, and was reversed by the kinase inhibitor staurosporine, strongly linking a mitotic kinase-phosphatase cycle with control of the assay. The histone kinase activity of the S-phase kinase p33cdc-2 did not promote transport inhibition, and destruction of p34cdc-2 a temperature sensitive cell line prevented the cyclin effect. These results supported the hypothesis that the mitotic kinase p34cdc-2 was responsible for transport inhibition, though probably not directly. Pharmacological and biochemical experiments suggested that the fusion of transport vesicles with their target was the site of the inhibition. The proteins involved are not known at present. These data support a model which links inhibition of vesicle fusion with the observed vesiculation of the Golgi during mitosis.

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