| Summary: | Cell division and the maintenance of cellular integrity are key features of life itself and some vital aspects of these processes have been studied in this thesis, using the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis as model eukaryotes.
In the first part of the thesis, three putative negative regulators of the cell wall integrity (CWI) signal transduction pathway were investigated, which have been isolated in previous genetic screens. Whereas the FIG4 gene seems to encode a protein which could be distantly related to CWI signaling, NTA1 and SET4 gene products were not found to have a major influence, as judged from phenotypes of deletion mutants,
overproducers, and epistasis analyses with different CWI pathway mutants. In general, the data indicated rather indirect connections of all three protein functions
with the maintenance of cellular integrity. Therefore, this line of research was
discontinued, in order to investigate more closely the regulation of cytokinesis in the
dairy yeast K. lactis.
In this second and major part of the thesis the homologue of a recently found
cytokinesis regulator in S. cerevisiae (INN1, accordingly designated as KlINN1), was
cloned and characterized. It could be shown, that the gene is essential and that the
encoded protein is species-specific, i.e. KlINN1 does not complement the lethality of
a Scinn1 deletion mutant and vice versa. Analyses of hybrid proteins demonstrated
that this specificity is most likely mediated by the C2-domain of the protein, which is thought to interact with membrane lipids. In S. cerevisiae, Inn1 interacts through its proline-rich motifs located in the C-terminal half of the protein with the cytokinesis regulators Hof1 and Cyk3. They both carry a SH3-domain which has been shown to mediate the interaction with Inn1. Consequently, the two encoding genes, KlHOF1
and KlCYK3, were also characterized in K. lactis. In contrast to S. cerevisiae, where
the homologues seem to exert somewhat redundant functions and only a double
deletion is lethal, each of the genes is essential in K. lactis. The exact nature of their roles in cytokinesis of this yeast remains to be determined. Unexpectedly, attempts to confirm an interaction between the proline-rich motifs of KlInn1 and the SH3-domains of KlHof1 and KlCyk3 in a yeast two-hybrid assay failed so far, but this line of research will be followed up in future experiments. In order to compare the timing of cytokinesis and the localization of the above mentioned regulators between S. cerevisiae and K. lactis, another homologue of a protein involved in cytokinesis in S. cerevisiae was investigated in K. lactis.
Preliminary evidence from deletion of the KlMYO1 gene, which encodes a likely
component of the contractile actomyosin ring (CAR) in K. lactis, indicates that this
yeast may predominantly engage in a CAR-independent pathway of cytokinesis.
Nevertheless, similar to S. cerevisiae GFP-fusions of KlInn1, KlHof1, KlCyk3, and
KlMyo1 all were shown here to localize to the bud neck during cytokinesis in K. lactis.
In summary, components identified to play a crucial role in yeast cytokinesis in
S. cerevisiae display a similar localization in K. lactis, but may differ considerably in
their detailed functions in vivo. This thesis represents the first detailed investigation of the molecular processes underlying cytokinesis in K. lactis and provides the basis for elaborate future studies.
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