UCSF

Posttranslational modifications enable cells to regulate their proteomes in a rapid and localized fashion. One such modification is ubiquitination and it is well conserved throughout eukaryotes and plays a role in nearly all intracellular processes. Ubiquitin is covalently attached to protein substrates through an enzymatic cascade in which ubiquitin ligases specify the target to be modified. The SCF superfamily of ubiquitin ligases are multisubunit enzymes that share a common scaffold which includes cullin (Cul1), an adaptor protein (Skp1) and F-box proteins that bind specific targets for degradation. To overcome limitations of current techniques to identify ubiquitinated substrates, we developed a technique in which ubiquitin-binding domains are fused to F-box proteins in order to enhance the stability of ligase-substrate interactions. These “Ligase Trap” constructs were then used to isolate polyubiquitinated substrates via a two-step purification procedure and the substrates identified by tandem mass spectrometry (Chapter 1). In budding yeast, Ligase Traps allowed us to identify both known and novel substrates using eight different F-box proteins. One F-box protein, Saf1, was found to target vacuolar/lysosomal zymogens (Chapter 2). Since it is unusual for a ligase to recognize an entire class of enzymes and because there is no known ubiquitin activity in a cell vacuole, I further examined how Saf1 interacts with its substrates (Chapter 3). My studies showed that SCFSaf1 specifically targets the catalytically active form of yeast vacuolar/lysosomal proteases. These results seem to suggest that ubiquitin ligases may interact with zymogens in the ER to regulate their activity or function prior to processing.