Functional analysis of cullin-based ubiquitin ligase and a novel UBC domain-containing protein

This thesis is composed of two major areas of study: the first study describes yeast two hybrid screen and biochemical approaches which were performed to identify interaction partners for C. elegans CUL-3; the second study focuses on the functional analysis of a novel ubiquitin conjugating (UBC) domain-containing protein FTS (Fused Toes). In the first study, a large family of proteins harboring a conserved BTB domain was identified to partner with Cul3. The BTB domain adopts a structure similar to the Cul1 interacting domain of Skp1. Most of the identified BTB domain proteins also contain an additional potential protein-protein interaction domain, such as MATH domain. We hypothesized based on analogy with the SCF complex, that BTB proteins function as substrate specific adaptors of the Cul3 Ub ligase. Mel-26, an essential MATH-BTB protein was one of the BTB proteins identified from the screen that interacts with CUL-3 in both yeast two hybrid and 293T transfection systems. Previous studies indicated that MEI-1, a microtubule-severing protein, is negatively regulated by MEL-26. We found MEL-26 interacts with MEI-1 through its N-terminal MATH domain and interacts with CUL-3 via its C-terminal BTB domain. We proposed that the BTB domain family of proteins merge the function and properties of Skp1 and F-box proteins into a single polypeptide, thereby acting as substrate specific adaptors for Cul3. In the second section of this thesis, FTS, a UBC domain containing protein of unknown function was found to interact with members of the microtubule-binding Hook family of coiled-coil proteins (Hook1, Hook2, and Hook3), and a previously uncharacterized 107 kDa protein we refer to as F TS and H ook I nteracting P rotein (p107 FHIP ). This study demonstrated that FTS uses residues in the β-sheet portion of its UBC domain to interact with a conserved sequence near the C-terminus of Hook proteins. FTS and Hook proteins interact with members of both the Class B and Class C components of the Ho motypic Vesicular P rotein S orting (HOPS) complex in human cells. Depletion of FTS, Hook1/2/3, or p107 FHIP by RNAi reduces the efficiency by which overexpression of the HOPS component Vps18 promotes clustering of LAMP1-positive endosomes. The trafficking of EGF from early endosomes to lysosomes is also affected in the presence of FTS small interference RNA. In summary, these data suggest that the FTS/Hook/p107 FHIP complex functions to promote vesicle trafficking and/or fusion via the HOPS complex.