Evolution and Divergence of the Genes for Cytoplasmic, Mitochondrial, and Flagellar Creatine Kinases Tomohiko Suzuki, 1 Chisa Mizuta, 1 Kouji Uda, 1 Keiko Ishida, 1 Kanae Mizuta, 1 Sona Sona, 2 Deanne M. Compaan, 2, * W. Ross Ellington 2 1 Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan 2 Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4370, USA Received: 16 December 2003 / Accepted: 10 February 2004 [Reviewing Editor: Martin Kreitman] Abstract. Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intra- cellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodi- mers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet an- other gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in proto- chordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a pro- tochordate tunicate. These new sequences, coupled with available sequences in the databases and se- quences extracted from genome sequencing projects, provide revealing insights into the evolution and di- vergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved af- ter the divergence of the craniates from the proto- chordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in struc- ture consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flag- ellar CK genes are rather ancient and that there has been a systematic pattern of duplication and diver- gence consistent with changing nature of energy de- mands and physicochemical environment in the cells where they are expressed. Key words: Creatine kinase — Isoforms Introduction Creatine kinase (CK) catalyzes the reversible transfer of phosphate from creatine phosphate to MgADP, yielding MgATP. This enzyme plays a central role in energy homeostasis in cells such as muscle fibers, neurons, transport epithelia, and spermatozoa (El- lington 2001). CK is a member of a highly conserved *Present address: Genentech, 1 DNA Way, South San Francisco, CA 1 94080, USA Correspondence to: W. Ross Ellington; email: elling@bio.fsu.edu J Mol Evol (2004) 59:218–226 DOI: 10.1007/s00239-004-2615-x