Purification and characterization of the mitochondrial translocase from Euglena gracilis

Abstract

The Euglena gracilis mitochondrial protein biosynthetic elongation factor G (EF-G mt) has been purified in four steps to greater than 50% homogeneity by use of a fusidic acid affinity procedure and conventional Chromatographic techniques. The purification scheme results in 1100-fold purification with about 3% recovery of the total EF-G activity present in the postribosomal supernatant prepared from whole cell extracts. E. gracilis EF-G mt has an approximate molecular weight of 76,000, comparable to that observed for procaryotic translocases. As is the case for other translocases which have been examined, pretreatment of E. gracilis EF-G mt with N-ethylmaleimide results in a loss of polymerization activity, indicating a role for an essential cysteine residue in catalytic activity. GDP partially protects EF-G mt from N-ethylmaleimide inactivation. E. gracilis EF-G mt functions well on both Escherichia coli and E. gracilis chloroplast ribosomes, but has negligible activity on wheat germ cytoplasmic ribosomes. In this respect, it differs significantly from the mitochondrial translocase of yeast which has very little activity on chloroplast ribosomes. When assayed on E. coli ribosomes, E. gracilis EF-G mt is sensitive to the steroid antibiotic, fusidic acid, at levels similar to that required for inactivation of E. coli EF-G. It is less sensitive than E. gracilis chloroplast EF-G, and is more sensitive than Bacillus subtilus EF-G. When assayed on E. gracilis chloroplast ribosomes, the same trends in sensitivities are observed, although the exact level of fusidic acid required for inactivation is slightly altered.