New non-lethal calmodulin mutations in Paramecium. A structural and functional bipartition hypothesis.
The mechanisms by which calmodulin coordinates its numerous molecular targets in living cells remain largely unknown. To further understand how this pivotal Ca(2+)-binding protein functions in vivo, we isolated and studied nine new Paramecium behavioral mutants defective in calmodulin. Nucleotide sequences of mutant calmodulin genes indicated single amino-acid substitutions in mutants cam4(E104K), cam5-1 (D95G), cam6 (A102V), cam7 (H135R), cam14-1 (G59S) and cam15 (D50G). In addition, we encountered a second occurrence of three identified substitutions; they are cam1-2 (S101F), cam5-2 (D95G) and cam14-2 (G59S). Most of these mutational changes occurred in sites that have been highly conserved throughout evolution. Furthermore, most of these changes were not among the amino acids known to interact with the basic amphiphilic peptides of calmodulin targets. Consistent with our previous finding [Kink, J. A., Maley, M. E., Preston R. R., Ling, K.-Y., Wallen-Friedman, M. A., Saimi, Y. & Kung, C. (1990) Cell 62, 165-174], mutants that under-reacted to certain stimuli (allele number above 10) had substitutions in the N-terminal lobe of calmodulin, and those that over-reacted (below 10) had substitutions in the C-terminal lobe. No mutations were found in the central helix that connects the lobes. Thus, through undirected in vivo mutation analyses of Paramecium, we discovered that each of the two lobes of calmodulin has a distinct role in regulating the function of a specific ion channel and eventually the behavior of Paramecium. We, therefore, propose a hypothesis of functional bipartition of calmodulin that reflects its structural bipartition.
Ling
K Y
KY
Laboratory of Molecular Biology, University of Wisconsin-Madison.
Maley
M E
ME
Preston
R R
RR
Saimi
Y
Y
Kung
C
C
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D017124:Conserved Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D002147:Calmodulin; D003062:Codon; D017931:DNA Primers