How to Catch Moby Dick: Systematic Identification of Binding Partners for UNC-89 (Obscurin)

Mutations in unc-89 result in adult C. elegans with reduced motility and disorganized, thinner sarcomeres. UNC-89-B (8,081 residues) has 53 Ig domains, 2 Fn3 domains, SH3, DH and PH domains at its N-terminus, and 2 protein kinase domains (PK1 and PK2) at its C-terminus. The human homolog is called “obscurin”. Antibodies localize UNC-89 to the M-line. To understand how UNC-89 is localized and how it performs its functions, we are systematically identifying its binding partners. UNC-89-B is entirely represented as 16 overlapping segments in 2-hybrid vectors. All 16 have been used to screen ∼23 known components of the nematode M-line. 7/16 have been used to screen a 2-hybrid library. We have identified 6 partners, each with human homologs, and all but one localized to M-lines. Both PK1 and PK2 interact with SCPL-1, a CTD-type protein phosphatase. scpl-1 mutants display defective egg laying muscles, and “hyper-bending” during locomotion. PK1 and the “interkinase region” interact with LIM-9 (FHL). lim-9(RNAi) show aggregates of myosin. Ig50-Ig51 interacts with HIF-1 (hypoxia inducible factor), and Fn1-Ig52 interacts with HUM-6 (class VII myosin). Ig1-Ig2-Ig3 interacts with CPNA-1, a copine domain containing protein. Loss of function for cpna-1 results in a Pat embryonic lethal phenotype, characteristic of mutations in 19 other genes, most of which encode products associated with integrin associated muscle adhesions. Three segments, Ig9-Ig11, Ig18-Ig23, and Ig50-Ig51 interact with CUL-1, a cullin, known to act as a scaffold for assembly of the ubiquitylation protein degradation machinery. cul-1(RNAi) shows disorganization of thick filaments in a pattern similar to that of unc-89(su75), an allele lacking all CUL-1 binding sites. My talk will focus on the role of CPNA-1 in localizing UNC-89, and the UNC-89/CUL-1 interaction as a novel means of regulating protein degradation in muscle.