Correlation of aggregation phenotypes and activity of mutant Ac transposase derivatives
--Manfred Heinlein, Sandra Kuehn, Ute Behrens and Reinhard Kunze

In a previous publication it has been reported that some inactive TPase mutants are dominantly inhibiting the transpositionally active TPase. Since two of these mutants are DNA-binding deficient, it has been proposed that the TPase acts as an oligomer or multimer during the excision reaction (Kunze et al., PNAS 87:7094-7098, 1993). We are interested to know whether the oligomerization of the TPase during the excision reaction and the tendency of the protein to form aggregates in vivo (see previous report) are related. Therefore, we started to look for correlations between the activity and the aggregation phenotype of various TPase mutants expressed in transfected petunia protoplasts.

Our tentative results indicate that 1) mutant TPase derivatives which have retained their activity also form aggregates identical to those formed by the wildtype protein and 2) that inactive TPase mutants are able to form aggregates if they are dominant. Both observations are consistent with the assumption that the ability to form aggregates is restricted to TPase molecules that also are able to form oligomers during the excision reaction.

One recessive and one dominant mutant give rise to a homogeneously distributed immunohistochemical signal. Obviously these mutants are not able to form aggregates, however, we also consider the possibility that the observed signal is due to aggregation intermediates. The fact that one of these mutants is dominant could suggest that the formation of TPase oligomers during the excision reaction and the aggregation of the protein occur via different pathways. However, it could also indicate that the protein-protein interactions necessary for oligomerization are not sufficient for the protein to aggregate. According to this hypothesis, aggregation would require an additional protein interactive surface.

Five of the six recessive mutant proteins we investigated are not detectable immunohistochemically although expression in the transfected petunia cells could be shown by Western blot analysis. In addition, certain pairs of these recessive TPase mutants complement to wildtype activity. We are presently trying to find an explanation for this apparent discrepancy. 

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