Phosphorylation motifs in the nonhelical domains of myosin heavy chain and paramyosin may negatively regulate assembly in Caenorhabditis elegans striated muscle.
We are interested in mechanisms that establish and maintain the highly ordered contractile apparatus of striated muscle. The homologous proteins myosin and paramyosin are the major structural components of thick filaments in invertebrate animals. In Caenorhabditis elegans, both proteins contain a homologous, small nonhelical domain that is known to be phosphorylated in paramyosin. In this report, we show that a proposed phosphorylation motif (S_S_A), which is present in several copies in the nonhelical regions of both myosin and paramyosin, is highly conserved among nematodes. We used in vivo assays to examine the assembly properties of proteins in which one or more motifs were targeted by point mutagenesis or deletion. In all cases, expression of mutant proteins improved the phenotype of the corresponding null mutant animals, but produced variable structural defects, including birefringent aggregates in adults and abnormal localization in embryos. Point mutation, but not deletion, of the myosin A nonhelical tailpiece produced ectopic structures that appeared as masses of jumbled filaments by TEM. Antibody labeling showed that aggregates of either mutant protein did not recruit the endogenous version of the other. Analysis of mutant embryos lacking either paramyosin or myosin A (the essential isoform at the thick filament center) indicated that both wild-type proteins can independently localize and initiate assembly, although the structures produced are abnormal. Our results suggest that muscle cells actively restrict myosin and paramyosin assembly through phosphorylation of the S_S_A motifs and that each protein is regulated independently.
Hoppe
Pamela E
PE
Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008-5410, USA. pamela.hoppe@wmich.edu
Heustis
Ronald J
RJ
Flanagan
Kelly A
KA
Reedy
April R
AR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000378:metabolism* / Q000648:ultrastructure; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D054792:Muscle, Striated / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D014335:Tropomyosin; D018995:Myosin Heavy Chains