Tight control of APC/C-Cdh1Fzr activity is essential for progression through mitosis and establishment of the G1 phase. Rca1 is a nuclear protein that inhibits the APC/C-Cdh1Fzr complex during G2 to allow cyclin accumulation and subsequent entry into mitosis. In this thesis, a localisation study of Rca1 was performed revealing that a nuclear localisation sequence (NLS) and other domains in the protein mediate efficient nuclear accumulation. Besides its function in G2, Rca1 expression can promote S phase entry. Rca1 belongs to the family of F-box proteins that mediate substrate specificity in SCF (Skp-Cul1-F-box) ubiquitin ligases. Functional studies demonstrated that the F-box is crucial for S phase induction by Rca1, suggesting that Rca1 has a secondary function in an SCF complex. A major part of this thesis was devoted to characterise the putative SCF/Rca1 complex and its target proteins. In a yeast two-hybrid screen, the SCF subunit SkpA was identified as a binding partner of Rca1. Coimmunoprecipitation studies confirmed this interaction and indicated moreover that SkpA binding relies on the F-box in Rca1. Furthermore, endogenous Cul1 coprecipitated with Rca1 and this also in an F-box dependent manner. Altogether, these experiments demonstrated that Rca1 forms a complex with the SCF core subunits SkpA and Cul1. The SCF/Rca1 complex could promote S phase entry by degrading a negative regulator of the G1-S transition. Cyclin A/Cdk1 is a potent S phase inducer, but its activity is normally dampened by the CKI Rux, inhibitory phosphorylation by Wee and APC/C-Cdh1Fzr dependent degradation of Cyclin A. Protein levels of these S phase inhibitors were not altered by coexpression of Rca1 suggesting that these proteins are not targets of the SCF/Rca1 complex. Overexpression of Rca1 in larval salivary glands results in impaired endoreplication and accumulation of Cyclin A, Cyclin E and Cdk1. Expression profiling revealed that mitotic genes (e.g. Cyclin A/B, Cdk1) are normally downregulated in salivary glands, but ectopic Rca1 expression promotes transcription of these genes. In addition, qRT-PCR analysis showed elevated transcript levels of the E2F1 targets Rnr2 and PCNA, suggesting that Rca1 overexpression leads to increased E2F1 activity. Cyclin E is also a known E2F1 target and hence, this might explain the marked increase in Cyclin E transcript levels. Finally, the gene expression analysis indicated that components of the APC/C and its target Geminin were present in larval salivary glands, thus supporting the idea that rereplication in endoreplicating cells is controlled by Geminin and the APC/C-Cdh1Fzr complex
