Identification and functional characterization of protein components of the plant kinetochore complex
Zusammenfassung der Projektergebnisse
1. Studies on transgenic plants expressing fluorescently labelled AtCENH3 (the centromere variant of the chore nucleosomal histone H3) and subsequent immunolabelling of endogenous CENH3 revealed the first precise cell cycle 'window' for deposition of CENH3 to a eukaryotic centromere. In A. thaliana this is mid to late G2. Surpringly, the sister kinetochores proved to be spatially separated simultaneously, i.e. before the onset of mitosis (Lermontova et al. Plant Cell, 2006). AtCENH3 requires the C-terminal histone fold domain for centromere targeting and recognises also the diverged centromeric repeats of A. lyrata. 2. A highly conserved putative KP, the A. thaliana homologue of yeast CBF5 is essential for viability, but is not detectable at A. thaliana or V. faba centromeres. Rather it occurs within nucleoli and Cajal bodies. There it interacts with the Arabidopsis homologue of yeast NAF1 that together with CBF5 is involved in H/A CA box snoRNP biogenesis and apparently required for pseudouridinylation of various RNA species. Thus, AtCBF5 is essential for RNA processing rather than being a KP (Lermontova et al. Plant J., submitted). 3. Arabidopsis homologues of the yeast spindle checkpoint proteins Bub1, Bub 3.1, Bub 3.2 and Zw10 were cloned and shown to be expressed in mitotically active tissues. Homozygous T-DNA insertions with bub genes revealed no transcription of the respective genes. The bubl mutant showed slower growth, the double mutant bub 3.1/3.2 displayed no deviating phenotype. Transgenic fluorescently labeled proteins were expressed at the mRNA level but yielded no centromeric signals. Polyclonal antibodies against a peptide of AtBub 3 stained field bean but not A. thaliana centromeres, although on Western blot an Arabidopsis protein of the expected size was detectable (manuscript in preparation). 4. The transgenic fluorescently labelled KP Skp1 marked the nucleus and the cytoplasm, while the checkpoint protein Zw10 revealed no fluorescence at all. Because no centromeric loalization was detectable, both proteins were not further investigated.
Projektbezogene Publikationen (Auswahl)
- Lermontova, I., Schubert, V., Fuchs, J., Klarte, S., Macas, J., and Schubert, 1. (2006). Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain. Plant Cell 18,2443-2451.
