The sarcomere contains a number of extraordinarily large polypeptides (700,000 Da-4 MDa) composed of multiple Ig and Fn3 domains, and one or two protein kinase domains. In C. elegans, one of these giants is UNC-89 (obscurin in humans). The largest UNC-89 isoform (~900,000 Da) consists of 53 Ig domains, 2 Fn3 domains, a triplet of SH3, DH and PH domains, and two protein kinase domains. Antibodies localize UNC-89 to the M-line. To understand how UNC-89 is localized and performs its functions, we are taking a systematic approach to identifying its binding partners. When UNC-89 segment Ig1-Ig5 was used to screen a yeast 2-hybrid library we pulled out MEL-26. When MEL-26 was used to screen a collection of 2-hybrid clones spanning the entire UNC-89-B coding sequence, one more region showed interaction, 1/3 interkinase-Ig53-Fn2. Minimally to interact with MEL-26, Ig2-Ig3, and Ig53-Fn2, are required. MEL-26 is one type of "substrate recognition protein" (and contains a BTB domain) that interacts with cullin 3. Cullin 3 is one type of cullin, highly conserved proteins that act as a scaffold for assembly of the ubiquitination machinery. Anti-MEL-26 antibodies (kindly provided by Lionel Pintard) localize to muscle M-lines and I-bands. RNAi of
mel-26 or
cul-3 beginning at the L1 larval stage (to avoid embryonic lethality), resulted in adult animals with body wall muscle having disorganization of GFP::MHC-A, in a pattern very similar to that observed in
unc-89(
su75) loss of function. We next examined sarcomeric organization in the temperature sensitive allele of
mel-26,
ct61sb4, marked with the neuronal Unc,
unc-29. This strain,
mel-26(
ct61sb4)
unc-29(
e1072), was grown at the permissive temperature, and resulting adults were stained with anti-MHC A. These animals, but not
unc-29(
e1072) showed disorganization of thick filaments similar to that observed in
mel-26(RNAi). MEI-1 (katanin) is the target of MEL-26 in C. elegans embryos (Pintard et al. Nature 2003). As adults,
mei-1(
ct46), a dominant gain of function and ts allele, that encodes a protein that cannot bind MEL-26, also shows disorganization of thick filaments. All but one of the
unc-89 mutant alleles (~20 total) display a thinner sarcomeric region, which we speculate results from increased degradation of sarcomeric proteins. When UNC-89 is deficient, we would expect that more MEL-26 would be available to promote the degradation of the sarcomere. Therefore, we hypothesize that normally the interaction of UNC-89 with MEL-26 prevents the CUL-3/MEL-26 complex from promoting the ubiquitin mediated degradation of sarcomeric proteins.