Engineering Ca2+/calmodulin-mediated modulation of protein translocation by overlapping binding and signaling peptide sequences.
Protein translocation is used by cells to regulate protein activity in time and space. Synthetic systems have studied the effect of second messengers and exogenous chemicals on translocation, and have used translocation-based sensors to monitor unrelated pathways such as caspase activity. We have created a synthetic Ca2+-inducible protein using calmodulin binding peptides that selectively reveal nuclear localization and export signals in low Ca2+ (0 microM) and high Ca2+ (10 microM) environments, respectively. Experiments in live cells showed that our construct translocates between the nucleolus and plasma membrane with time constants of approximately 2 h. Further, a single amino acid mutation (Cys20Ala) in our construct prevented translocation to the plasma membrane and instead targeted it the mitochondria as predicted by bioinformatic analysis. Lastly, we studied the effect of cell line, Ca2+ concentration, chemical inhibitors, and cell morphology on translocation and found these conditions affected the rate, extent and direction of translocation. Our work demonstrates the feasibility of engineering Ca2+/calmodulin-mediated modulation of protein translocation and suggests that more natural analogs may exist.
Mills
Evan
E
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S3G9, Canada.
Truong
Kevin
K
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019295:Computational Biology; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000235:genetics; D009697:Nucleolus Organizer Region / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D011994:Recombinant Proteins