Dr. Neal Mathias' Research Focus

Major Research Interests:

The ubiquitin (Ub)-proteasome system is the major mechanism of protein degradation in all eukaryotes. It plays a critical role in diverse biological processes including the immune response, cell cycle progression, signal transduction, and transcription. The transfer of Ub to a substrate is a multi step process requiring E1, to activate Ub, and E2, to conjugate Ub to substrate. The role of E3, or ubiquitin ligases is to recognize specific substrates. One of the largest families of ubiquitin E3 ligases is the evolutionarily conserved multimeric complex referred to as the SCF family. SCF complexes contain a common set of components, namely, Skp1p, Cdc53p, Rub1p, and the RING finger protein Hrt1p. A variable component, called the F-box protein, is responsible for substrate recognition. Through the F-box motif-Skp1p interaction, the F-box protein is tethered to SCF core components. Through a second protein-binding motif on the F-box protein, substrate selection is achieved. We have exploited the genetic and biochemical tractability of the budding yeast Saccharomyces cerevisiae to probe the mechanisms of SCF complex biogenesis, substrate selection, and regulation. As part of the SCF^Met30p complex, Met30p selects the substrate Met4p, a transcriptional activator for MET biosynthetic genes that mediate sulfur uptake and biosynthesis of sulfur containing compounds. The accumulation of intracellular cysteine, one of the end products of the MET biosynthetic pathway, represses MET gene expression by signaling SCF^Met30p-mediated ubiquitination of Met4p. The focus of our laboratory is to determine the regulatory mechanisms of Met4p ubiquitination and that of SCF^Met30p biogenesis. Met30p is a nuclear protein and we have determined that the entire amino terminus of Met30p, including its F-box motif, is required for its proper nuclear localization. However, Skp1p is not required for Met30p nuclear localization. Also, sequences within the amino terminus of Met30p are important for its assimilation into an SCF complex. In addition to the F-box motif, which directly contacts Skp1p, we have observed that mutations upstream of the F-box motif are important for Skp1p binding. Thus, we propose that proteins binding to the amino terminal region of Met30p play a critical role in SCFMet30p biogenesis. The carboxyl terminal region of Met30p is composed almost entirely of seven WD 40 repeats. We have demonstrated that these are critical for contacting Met4p and have identified residues important for this interaction. Finally, we have reconstituted SCFMet30p ubiquitin ligase activity using recombinant proteins generated in insect cells. These tools are being exploited to determine how the ubiquitination of Met4p is regulated.