Functional Expression of Human Adenine Nucleotide Translocase 4 in Saccharomyces Cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells

Missense translation errors in Saccharomyces cerevisiae

We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CAT(III)) expressed in S. cerevisiae. A His195(CAC) --> Tyr195(UAC) mutant of CAT(III) is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon. The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0.5 x 10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic. A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2 x 10(-5), but in contrast to the Tyr195 --> His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction. The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants. These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44(+) and sup46(+) genes encode the yeast ribosomal proteins S4 and S9, respectively. These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions