We have reconstituted the fragmentation and dispersal of Golgi membranes by incubation of permeabilized Normal Rat Kidney (NRK) cells with mitotic cytosol and an ATP regenerating system at 32 C. We find that mitotic cytosol depleted of p34cdc2 kinase (the kinase necessary for entry into mitosis) causes Golgi fragmentation. Depletion or inactivation of the cytosolic Mitogen activated protein kinase kinase (MEK1), however, inhibits Golgi fragmentation. Adding back MEK1 to MEK1 depleted mitotic cytosol restores Golgi fragmentation. We find that the downstream (and the relevant) cytosolic MAPkinase, ERK1 and ERK2 are not required for this process. Interestingly, a MAPkinase, recognized by an antibody with broad cross-reactivity to ERK2 is tightly associated with Golgi membranes. Our overall goals for this proposal are to understand the mechanism by which MEK1 is activated and test our hypothesis that Golgi MAPkinase is the immediate downstream target of activated MEK1. Our studies should reveal the identity of a regulator termed MEK1-kinase necessary for MEK1 activation, components that are involved in dispersal of the fragmented Golgi membranes and finally the Golgi membrane associated proteins, including the MEK1 substrate required Golgi fragmentation. These studies should provide an under- standing of the mechanism by which Golgi membranes undergo extensive fragmentation at the onset of mitosis.
|Guizzunti, Gianni; Brady, Thomas P; Malhotra, Vivek et al. (2007) Trifunctional norrisolide probes for the study of Golgi vesiculation. Bioorg Med Chem Lett 17:320-5|