EUKARYOTIC CELL CYCLE REGULATION

The main interest of our laboratory is to understand the role of ubiquitin-dependent proteolysis in the control of the eukaryotic cell cycle. We exploit the genetic, molecular and cell biological techniques available in Drosophila melanogaster for mutant isolation and functional analysis of cell cycle regulation and ubiquitylation.

Ubiquitin-dependent protein degradation in Drosophila

Orderly and timely progression through the cell division cycle is essential to maintain genome stability in eukaryotes. Errors in this process alter gene dosage and can result in developmental defects or cancer. Key transitions in the eukaryotic cell cycle are regulated through intracellular proteolysis by the ubiquitin-proteasome system (UPS). Due to the irreversible nature of protein degradation, by sequentially removing regulators, this process generates directionality in the cell cycle and ensures proper timing and coordination of events.

Degradation of proteins by the UPS involves two successive steps: tagging of substrate proteins by the covalent attachment of polyubiquitin chains and the subsequent degradation of the tagged protein by the 26S proteasome. Besides the ubiquitin conjugating pathway and the 26S proteasome, additional factors like deubiquitylating enzymes (DUBs) and polyubiquitin binding proteins (UBPs) are involved in the efficient degradation of target proteins.

The ubiquitin conjugating pathway involves an enzymatic cascade of ubiquitin-activating (E1), ubiquitin-conjugating (E2), ubiquitin ligase (E3) enzymes, and at least sometimes, specific polyubiquitin chain elongation factors (E4), which catalyze the assembly of polyubiquitin chains on lysine residues of substrate proteins. The key component of this protein degradation machinery is the ubiquitin ligase, because it determines substrate specificity.

Two critical transitions in mitosis, the metaphase to anaphase transition and the exit from mitosis are mediated by a large ubiquitin-protein ligase complex known as the anaphase-promoting complex or APC. The APC is composed of at least twelve subunits; most of them are evolutionally conserved in all eukaryotes from yeasts to humans.

Though the main function of APC and key steps in its regulation are well established, many important questions related to the ubiquitylation state and function of regulatory proteins remain unanswered. We don't yet know why so many subunits are present in the APC or what their individual functions are. It is unclear how the APC mediates the assembly of polyubiquitin chains on substrates and how these tagged proteins are escorted and recognized by the 26S proteasome. Little is known about the regulatory role of deubiquitylating enzymes (DUBs) that remove ubiquitin molecules from proteins.

Our group is focusing on studying the role of APC subunits, DUBs and UBPs in the proteasomal degradation of mitotic regulatory proteins in the fruit fly, Drosophila melanogaster. We use P element-based forward and reverse genetic techniques to isolate mutant alleles of genes involved in these processes and to determine their roles.