Molecular structure and functional characterization of arginine kinase isoforms in Drosophila melanogaster.

摘要

The enzyme arginine kinase is a phosphotransferase that transfers the high energy phosphate of ATP onto arginine and synthesizes the cellular energy buffer molecule, phosphoarginine, in the fruit fly, Drosophila melanogaster and other invertebrates. The arginine kinase/phosphoarginine ATP buffering system maintains ATP homeostasis during high and fluctuating energy demands of the cell. The arginine kinase gene, Argk, of Drosophila melanogaster spans 17.144 kilobases (kb) and comprises of 7 exons and 6 introns. Annotation of the completely sequenced Drosophila genome revealed six putative alternative transcripts ( ArgkRA, ArgkRB, ArgkRC, ArgkRD, ArgkRE and ArgkRF) from the single Argk gene. All six forms share a large, common C-terminal catalytic domain and have small, variable and unique N-terminal domains. In this work I studied the distribution of the alternative transcripts in different tissues of the fly. I obtained partial cDNA of five alternative transcripts from total RNA of different tissues by RT-PCR using transcript specific primers. The transcripts appear to be widely distributed compared to their corresponding proteins separated based on size and detected on an immunoblot. Two of the corresponding proteins products are found in the thorax and constitute the bulk of activity present in the adult fly. The protein products of the remaining transcripts are largely restricted to the testis and ovary. I have demonstrated for the first time that one of the transcripts (RA) is a novel nuclear-encoded arginine kinase isoform (mtAK, 66kDa) from Drosophila melanogaster that is targeted for import into mitochondria. Live cell imaging analyses of mtAK, its distinct N-terminal region and a series of N-terminal deletions fused to the green fluorescent protein (GFP) identified a signal peptide of 21 amino acids that is responsible for targeting the protein into the mitochondria of cultured Drosophila S2 cells. The signal peptide, located 13 amino acids away from the N-terminal methionine, is located at the beginning of the unique proline-rich domain of the isoform, and appears to be a non-cleavable targeting signal for import of mtAK into the mitochondria. This is the first instance of compartmentalized protein isoforms of a phosphagen kinase being the products of alternative transcripts of a single gene.