Upregulation of the mitochondrial phosphate carrier during freezing in the wood frog Rana sylvatica: potential roles of transporters in freeze tolerance.

Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica) identified a freeze-responsive clone containing a 1370-nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over a time course of freezing, reaching 60-fold upregulation after 24-h frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase in pic transcript levels in response to dehydration (elevated 9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers (dicarboxylate carrier, oxoglutarate transporter) of the same family were not affected by freezing. This modulation of PiC protein levels may play a role in mitochondrial ionic and/or osmotic balance during freeze-induced cell volume reduction.