A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.

The ubiquitin-like protein Nedd8/Rub1 covalently modifies and activates cullin ubiquitin ligases. However, the repertoire of Nedd8-modified proteins and the regulation of protein neddylation status are not clear. The cysteine protease DEN1/NEDP1 specifically processes the Nedd8 precursor and has been suggested to deconjugate Nedd8 from cullin proteins. By characterizing the Drosophila DEN1 protein and DEN1 null (DEN1null) mutants, we provide in vitro and in vivo evidence that DEN1, in addition to processing Nedd8, deneddylates many cellular proteins. Although purified DEN1 protein efficiently deneddylates the Nedd8-conjugated cullin proteins Cul1 and Cul3, neddylated Cul1 and Cul3 protein levels are not enhanced in DEN1null. Strikingly, many cellular proteins are highly neddylated in DEN1 mutants and are deneddylated by purified DEN1 protein. DEN1 deneddylation activity is distinct from that of the cullin-deneddylating CSN. Genetic analyses indicate that a balance between neddylation and deneddylation maintained by DEN1 is crucial for animal viability.

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