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Basic analysis of cell cycle genes

The SCF complex is a protein degradation machinery that consists of at least three different types of proteins, being SKP1, cullin and F-box protein. In contrast to yeast that encodes single SKP1 and cullin/CDC53 SCF subunits and only a few F-box proteins, the Arabidopsis genome contains 19 SKP1, 6 CULLIN and several hundreds of potential F-box protein genes. This result was totally unexpected and a first and major objective of this study is therefore to identify those members of the SKP1, cullin and F-box protein families that truly have a function in cell cycle control.

Dudits' group has searched for those F-box genes that are expressed in a cell cycle-dependent fashion, as this is indicative of a role in the cell division process. For this purpose, construction of an Arabidopsis F-box gene array was initiated. Based on the alignment of all Arabidopsis F-box proteins and by similarity searches, further 22 F-box cDNAs were cloned. To generate probes for array hybridisation, an Arabidopsis (ecotype Ler) cell culture (obtained from L.Bogre) was synchronised successfully by aphidicolin block-release. RNA samples prepared from cells in different cell cycle phases were then converted to labelled cDNA via reverse transcription.

A second approach followed by Dudits' group aimed at investigating the substrates of F-box proteins, as it is anticipated that F-box proteins with a role in cell cycle control would have known cell cycle proteins as substrates. One strategy to identify such substrates is through 2-hybrid screens in yeast. It should be noted however that substrate proteins of F-box proteins are post-translationally modified before being degraded by these F-box proteins. Since it is unclear whether interaction between substrate and F-box protein occurs without such modifications and since it is unknown whether these modifications would occur in yeast, applying a 2-hybrid screen for identifying potential substrates of F-box proteins is an approach of which the success is unpredictable. So far, this approach did not yield any known cell cycle proteins as interactors. Yet, the 2-hybrid screen was experimentally validated by the fact that one of the identified interactors was an SKP1 protein, a known partner of F-box proteins in the SCF complex.

An alternative strategy for looking at substrates of F-box proteins has also been initiated. This approach should be more reliable than the yeast 2-hybrid screen as it looks for protein interactions directly in plants. Yet, it is at the same time more laborious and technically more demanding. Wild type and truncated (F-box deleted) versions of F-box proteins were fused to HA or Myc epitopes and introduced into Arabidopsis cells via Agrobacterium-mediated transformation. Expression of epitope-tagged proteins was analysed by western blotting. From transformed cell lines protein extracts were prepared and after a prefractionation step complexes were purified by immunoaffinity chromatography. Purified complexes were fractionated further via two-dimensional gel electrophoresis and subunits are currently being identified by MALDI-TOF spectrometry. At the same time, these constructs have been transformed into transgenic plants, in order to investigate the phenotypic effects of over-expression of specific F-box proteins on overall plant development.

The interaction of FB2 with Arabidopsis D-type cyclins 3.2; 4.1 and 5.1 suggests that FB2-SCF complexes might have a role in the regulation of CDK kinase activity by controlling cyclin stability. The interaction between FB10 and two different RING proteins is confirmed. The C terminal CUE domain, thought to bind Ub-conjugating enzymes is indeed required for binding. Its HRD1-RING-CUE domain structure is similar to that of human gp78 tumor autocrine motility factor receptor that is involved in ER associated degradation of proteins confirming the role of the RING finger candidate genes in protein degradation.

A collaboration was started between Murray's group and the lab of P Genschick in Strasbourg, France to study one particular RING finger involved in the SCF complex.

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Biological Research Centre of the Hungarian Academy of Sciences
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6726 Szeged
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