Gene expression supporting freeze tolerance in the wood frog, Rana sylvatica
Natural freezing survival by the wood frog, Rana sylvatica, is supported by multiple metabolic and gene expression adaptations. The present study explored the responses by protein chaperones, both heat shock proteins (Hsps) and glucose-regulated proteins (Grps), as well as the hypoxia-inducible transcription factor (HIF-1α), to freezing and two component stresses of freezing (anoxia, dehydration) in seven organs of wood frogs. Sequence analysis of Grp78, Grp94 and HIF-1α found high identity between the frog proteins and other species but HIF-1α showed unique amino acid substitutions that might aid low temperature function. RT-PCR and Western blotting were used to monitor mRNA transcript levels and protein levels, respectively. All three stresses stimulated up-regulation of HIF-1α at transcriptional and translational levels in liver. Other organs showed stress-specific responses and time course studies showed maximum hif-1α mRNA levels by 2 hours of freezing which suggests that hypoxia-induced proteins may contribute tocryoprotection. Levels of all seven shock proteins (Grp78, Grp94, Hsp110, Hsc70, Hsp60, Hsp40, Hsp10) rose during freezing in liver; six responded in skeletal muscle and kidney, and four in heart. The distribution of shock proteins and HIF-1α between liver cytosolic and nuclear fractions also changed during freezing. Most shock proteins responded to anoxia stress in liver and to dehydration stress in skin but other organs showed mixed responses to these stresses. The mitochondrial chaperones Hsp60 and Hsp10 were consistently elevated during freezing and/or anoxia in most organs. Up-regulation of chaperones during freezing suggests that they play a cryoprotective role in stabilizing proteins against the multiple stresses on cells imposed by extracellular freezing.