Abstract
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.
Highlights
The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane
The idea was to select transformants based on their ability to grow on media requiring a functional mitochondrial respiratory system by providing sufficient adenine nucleotide transport activity to support growth of yeast on nonfermentable carbon sources as described previously [10]
Several previous reports as well as data presented here demonstrated that the N-terminal sequence greatly influences the functional localization of ANT proteins [15]
Summary
The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. Proper function of ANT is essential for the transfer of ATP synthesized in mitochondria to the cytoplasm. Most eukaryotes from yeast to humans have multiple ANT isoforms [1]. Unicellular organisms utilize different ANT isoforms depending on the availability of external nutrients and aeration. Multicellular organisms, express different ANT isoforms in a tissue-specific manner that are apparently adapted to the unique metabolic demands of the various tissues. The previous gene knock-out study in mouse revealed that ANT4 was essential for the process of male germ cell meiosis in mice [6], neither the function of ANT4 in male germ cells nor the reason why ANT4 exists only within a limited spectrum of species is known
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