Calmodulins with deletions in the central helix functionally replace the native protein in yeast cells.
Deletion of Glu-84, Glu-83 and Glu-84, or Ser-Glu-Glu-Glu (residues 81-84) from the central helix of mammalian calmodulin is known to result in a 5-7 times decrease in its apparent in vitro affinities for three calmodulin-dependent enzymes. However, based on in vitro experiments, it is difficult to estimate how these deletions might affect in vivo cellular function. The yeast Saccharomyces cerevisiae, which requires calmodulin for growth, provides an excellent system to evaluate these deletion proteins in vivo. Based on its ability to restore normal growth characteristics to yeast cells, mammalian calmodulin is functionally identical to the yeast protein; herein we evaluate the effect of deleting residues 84, 83 and 84, or 81-84 from the central helix. Sequences encoding the deletion proteins and an unaltered control sequence were introduced by means of a yeast shuttle vector and were expressed under control of the yeast calmodulin promoter. The deletion and control calmodulins are produced at levels similar to that observed for the yeast protein, and they completely restore normal growth characteristics. This result suggests that the regions deleted from the central helix are not critical for activation of any yeast calmodulin target normally required for cell growth or division. It is likely that there are twisting and shortening motions associated with the deletions from the central helix that alter significantly the spatial relationship between the two lobes of calmodulin. The abilities of the deletion calmodulins to restore completely normal growth characteristics to yeast cells suggest that the lobes of all the deletion proteins can still be appropriately positioned in calmodulin-target complexes. This is consistent with the hypothesis that the central helix of calmodulin is analogous to a flexible tether rather than to a rigid connector between the two lobes of the molecule.
Persechini
A
A
Department of Physiology, University of Rochester School of Medicine and Dentistry, NY 14642.
Kretsinger
R H
RH
Davis
T N
TN
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002872:Chromosome Deletion; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D012689:Sequence Homology, Nucleic Acid
D002147:Calmodulin