The ultimate aim of this work was to pinpoint a transport event or protein which was affected by mitosis. Initially, that was done by seeing whether membranes or cytosol were more affected. It was not feasible to isolate mitotic membranes, so they were always supplied to the assay in an 'interphase' state. The fact that mitotic or cyclin A-activated cytosols inhibited K-Golgi membranes more strongly than unwashed membranes suggested that a cytosol- derived factor was the primary target of the mitotic cytosol. K-Golgi is known to be stripped of a number of proteins which normally cycle on and off the membranes (Weidman et al,
1989), and it was an obvious suggestion that interphase proteins were removed by salt- washing to make way for the 'inactivated' mitotic ones.
This indication was supported by an experiment in which 'arrested' mitotic cytosol could be supplied with transport proteins by interphase cytosol. The simplest interpretation of that result was that transport components of cytosol had been inactivated by mitotic
phosphorylation, thus immobilising an otherwise complete transport system. Then, addition of interphase cytosol resupplied the immobilised components, and transport could proceed. Given the attractive model in which inhibition of transport vesicle fusion leads to Golgi vésiculation, it would be satisfying if acceptor function were inhibited by mitotic kinase activity, leading to accumulation of vesicles in mitotic cytosol. It is also possible, however, that Golgi breakdown is conducted by an entirely separate process, and inhibition of vesicle fusion simply ensures against inappropriate vesicle mixing at any point in the entry to or exit from mitosis.
In one approach to this problem, donors or acceptors were preincubated in mitotic cytosol, and then arrested and added to interphase assays with the interphase-treated membranes. In this system, it was found that acceptors incubated in mitotic cytosol were less active in a complete assay than mitotic-treated donors. The assay was handicapped by the marked loss of counts in any form of assay where donors and acceptors were incubated separately under transport conditions.
Transport inhibition could also be studied by using inhibitors of various stages of transport. The agents mastoparan and aluminum fluoride both act on trimeric G-proteins, which have been localised to the Golgi and implicated in control of constitutive transport. Both of these had equal effects on interphase and mitotic assays, though in the case of mastoparan the effect was so extreme that it seemed to be due to a detergent action rather than influence on transport Brefeldin A, which prevents assembly of COP-coated vesicles and causes
promiscuous membrane fusion, increased apparent transport in interphase assays, but not mitotic ones. This suggested that fusion was inhibited in the mitotic assay at a post-uncoating stage, but it could not be ruled out that budding was profoundly inhibited.
GTP7S inhibited mitotic and interphase transport assays to the same extent Bearing in mind
that salt-washed and unwashed transport were also equally affected by the drug, this result strongly suggested that coated vesicles were both formed and uncoated in interphase and mitotic cytosol, ie that the stage inhibited in mitosis was after the uncoating of the transport vesicles. Taken together with the effect of brefeldin, this experiment places the site of
inhibition at a point after vesicle uncoating but before vesicle fusion. However, in interphase assays the effect of GTPyS was almost nil after 20 minutes, whereas in mitotic cytsol the drug still inhibited transport at that time. This suggests that a stage before vesicle fusion was also affected by mitotic conditions. Overall, the results from experiments with transport inhibiting drugs did not unambigously indicate a site of mitotic transport inhibition.
Another way of examining the mechanism of transport inhibition was to guess that the target might be one of the fusion proteins described by the Rothman group, and test whether any of those could be implicated in inhibition or recovery from inhibition. SNAPs and NSF were added to the transport assay either singly or in combination. The assay was notably stimulated by a-SNAP and less so by y-SNAP or NSF, and this is in line with results of Rothman and co-workers. NSF is thought to act ’catalytically’ in the assay, and therefore is stimulatory at a much lower level then the two SNAPs. a-SNAP appeared to be limiting in sHeLa cytosol at the levels normally used in transport, but y-SNAP was not apparently limiting. Its role in fusion is not yet clear.
When these proteins were added to interphase or cyclin-treated cytosol, the assay was stimulated to just the same extent, whether or not it contained active phosphatases and
kinases. Thus, under those conditions, none of the proteins were apparently targets for cyclin- mediated inactivation. This is consistent with the result that cyclin treatment of cytosol is less effective than simultaneous treatment of membranes and cytosol; if cytosol treatment alone were very inhibitory, then it might be expected to act on proteins like SNAPs and NSF. It also suggested that these proteins were after the rate-limiting stage of transport in the cyclin- treated assay.
In contrast to cyclin-treated cytosol, mitotic cytosol did not support significant transport enhancement by SNAPs or NSF, in the presence or absence of kinase/phosphatase inhibitors. This is interesting since the proteins were added to the cytosol before the membranes but after any arrest of cytosol, ie the proteins were functional targets of mitotic inhibition, or downstream of it, and kinase activity on them or the membranes was not required.
The finding that SNAPs and NSF had different effects on cyclin A-treated interphase cytosol and mitotic cytosol was unexpected, considering how alike the two had been in previous experiments. It suggested that the two conditions inhibited transport in different ways. This difference between cyclin and mitotic cytosol was the only indication in this work that cyclins A and B regulated transport differently, but the difference could not be explored further using the transport assay alone due to the failure of cyclin B to activate sHeLa cytosoL It appeared that the assay could be inhibited in more than one way. It was also plain that recombinant SNAP and NSF could not rescue transport in arrested mitotic cytosol. The simple idea that phosphorylation of a cycling fusion protein like NSF or SNAP inhibited the assay had to be abandoned.
Electron micrographs of mitotic and interphase transport assay mixtures (containing unwashed Golgi) did not show up any clear differences between them (T. Misteli, personal communication); perhaps this was due to the relatively disreputable appearance of CHO- derived Golgi membranes, or due to weak inhibition of transport in the reactions selected. However, rat liver Golgi (which can rapidly be purified over 200-fold) incubated with mitotic cytosol in BBS buffer (in which cytosol is made) sheds vesicles at a rate comparable with the published calculated rates of vesicle producion by in-vitro systems (T. Misteli, manuscript in preparation). It will be extremely interesting to see whether the mechanism, rate and extent of vesicle shedding are comparable to those seen in transport