Meeting Abstract
P1.112 Sunday, Jan. 4 Cytoplasmic and Mitochondrial Arginine Kinase Isoforms in a Choanoflagellate Protozoan CONEJO, M.S.; HOFFMAN, G.G.*; ELLINGTON, W.R.; Florida State Univ. wellington@admin.fsu.edu
Arginine kinase (AK) catalyzes the reversible transfer of phosphate from phosphoarginine to MgADP yielding MgATP and arginine. This enzyme plays a central role in mitigating temporal and spatial ATP supply and demand mismatches in cells displaying high and variable rates of ATP turnover. AK is widely distributed in invertebrates and protochordates but is lacking in craniates. A considerable base of molecular phylogenetic data and other evidence support the view that choanoflagellate protozoans are a direct sister group of the metazoans. The recently released genomic and EST sequence database for the choanoflagellate Monosiga brevicollis contains clear-cut evidence for at least three AK genes in this protozoan. We have generated the cDNAs for two of these by RTPCR. One corresponds to a 394 residue AK with a calculated Mr of 42.57 kDa. This protein displays great amino acid similarity to that of an AK from Aphrocallistes beatrix, a hexactinellid sponge. On-line tools predicted that this AK is targeted to the cytoplasm (cytAK). Expression of the cytAK in E. coli yielded highly active soluble protein which was shown to be monomeric by size exclusion chromatography. The other AK cDNA coded for a 421 residue protein with a calculated Mr of 45.71 kDa. On-line tools indicated that this AK has an N-terminal signal peptide. The majority of the tools predicted that this AK is targeted to the mitochondrion suggesting that it is a true mitochondrial AK (MiAK). Efforts are underway to express various MiAK constructs to assess activity and quaternary structure. The present results support the view that cytoplasmic and mitochondrial isoforms of AK are expressed in M. brevicollis. These AKs very likely serve to mitigate reaction diffusion constraints for energy transport between mitochondria in the cell body and the long flagellum used in motility and food entrapment. (Supported by NSF Grant IOS 0542236 to WRE).
