The Caenorhabditis elegans gene unc-89, required for muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

Abstract

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines (Waterston, R.H., J.N. Thomson, and S. Brenner. 1980. Dev. Biol. 77:271-302). Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in-frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632-amino acid polypeptide consisting of sequence motifs which have been implicated in protein protein interactions. UNC-89 begins with 67 residues of unique sequence, SH3, db1/CDC24, and PH domains, 7 immunoglobulin (Ig) domains, a putative KSP- containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane (Williams, B.D., and R.H. Waterston. 1994. J. Cell Biol. 124:475-490; Hresko, M.C., B.D. Williams, and R.H. Waterston. 1994. J. Cell Biol. 124:491-506). We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line.