The dramatic change in Golgi morphology during mitosis in animal cells is part of the process that ensures inheritance of this organelle through successive generations. During prophase, the Golgi ribbon breaks down into dispersed stacks which fragment into clusters of vesicles and tubules. During telophase this process is reversed and a Golgi ribbon is formed in each daughter cell. Using a cell-free assay that reconstitutes many aspects of this process we have been able to identify NSF and p97 as two of the ATPases needed to reassemble cisternae from mitotic Golgi fragments. Both ATPases act through the Golgi t-SNARE, syntaxin 5, to rebuild Golgi cisternae, but the underlying mechanism is different.
The aim of this project is to work out precisely how these two ATPases operate and co-operate to rebuild Golgi cisternae and how this process is regulated during mitosis.
The specific aims are: 1. To work out the mechanism of action of p97. To test the hypothesis that p97 breaks up t-t SNARE complexes just as NSF breaks up v-t SNARE complexes. We will identify precisely which Golgi SNARES are involved and which other components are needed to separate and prime the SNARES. Eventually we will reconstitute this process using recombinant and purified proteins incorporated into liposomes. 2. To study the mitotic regulation of Syntaxin 5 and p97. We will build on our preliminary observations that mitotic syntaxin 5 is part of a new complex. We will map the phosphorylation sites that permit incorporation into this complex and identify the other proteins in it. We will explore the idea that inactivation of p97 during mitosis is caused by phosphorylation of p47 and consequent dissociation from p97. 3. To elucidate the respective roles played by p97 and NSF in the reassembly of the Golgi. We will test the idea that p97 assembles the cores whereas NSF assembles the rims using our cell-free assay and micro-injection into mitotic cells. We will determine whether p97 can stitch Golgi stacks together to give a polarised ribbon and analyse the consequences of disrupting this ribbon in vivo.
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