Differential posttranslational modification of mitochondrial enzymes corresponds with metabolic suppression during hibernation

Abstract

During hibernation, small mammals, including the 13-lined ground squirrel (Ictidomys tridecemlineatus), cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5°C, and interbout euthermia (IBE), where both metabolic rate and body temperature rapidly increase to euthermic levels. Suppression of whole animal metabolism during torpor is paralleled by rapid, reversible suppression of mitochondrial respiration. We hypothesized that these changes in mitochondrial metabolism are regulated by posttranslational modifications to mitochondrial proteins. Differential two-dimensional gel electrophoresis and two-dimensional blue-native PAGE revealed differences in the isoelectric point of several liver mitochondrial proteins between torpor and IBE. Quadrupole time-of-flight LC/MS and matrix-assisted laser desorption/ionization MS identified these as proteins involved in β-oxidation, the tricarboxylic acid cycle, reactive oxygen species detoxification, and the electron transport system (ETS). Immunoblots revealed that subunit 1 of ETS complex IV was acetylated during torpor but not IBE. Phosphoprotein staining revealed significantly greater phosphorylation of succinyl-CoA ligase and the flavoprotein subunit of ETS complex II in IBE than torpor. In addition, the 75-kDa subunit of ETS complex I was 1.5-fold more phosphorylated in torpor. In vitro treatment with alkaline phosphatase increased the maximal activity of complex I from liver mitochondria isolated from torpid, but not IBE, animals. By contrast, phosphatase treatment decreased complex II activity in IBE but not torpor. These findings suggest that the rapid changes in mitochondrial metabolism in hibernators are mediated by posttranslational modifications of key metabolic enzymes, perhaps by intramitochondrial kinases and deacetylases.

Keywords: acetylation; metabolic suppression; mitochondrial metabolism; phosphorylation; succinate dehydrogenase.

Fig. 1.

Ground squirrel liver mitochondrial proteins in torpor and interbout euthermia. Representative 2-dimensional differential gel electrophoresis gel shows liver mitochondrial protein spots from 1 torpid (Cy3, labeled green) and 1 interbout euthermia (Cy5, labeled red) animal. Proteins were separated by isoelectric point (pI; horizontal dimension) and then by molecular mass (vertical dimension). Arrows indicate proteins of interest, which are summarized in Table 1.

Fig. 2.

Representative gels showing phosphoprotein staining of liver mitochondrial protein from 1 ground squirrel in interbout euthermia (IBE, A) and 1 ground squirrel in torpor (B) separated by 2-dimensional blue-native PAGE. Liver mitochondrial protein was separated by molecular mass into individual electron transport system complexes in the 1st dimension (horizontal) under nondenaturing conditions. Protein was then separated by molecular mass again in a 2nd dimension (vertical) under denaturing conditions using SDS-PAGE. Arrows indicate proteins that differed significantly in phosphorylation between torpor and IBE (4 each in torpor and IBE), with corresponding spots indicated by the same number on each gel. Spot 22 is present in IBE (A) but is not visible in torpor (B); an arrow indicates where the spot would be if present. Data from all blue-native PAGE gels are summarized in Table 2.

Fig. 3.

Representative 2-dimensional blue-native (BN) PAGE gels for lysine acetylation in liver mitochondrial protein from 1 ground squirrel at interbout arousal (A) and 1 ground squirrel in torpor (B). Liver mitochondrial protein was separated by molecular mass into individual electron transport system complexes in the 1st dimension (horizontal) under nondenaturing conditions and again in a second dimension (vertical) under denaturing conditions using SDS-PAGE. Arrows indicate proteins that differ significantly in acetylation between torpor and IBE (4 each in torpor and IBE), with corresponding spots indicated in each gel.

Fig. 4.

Maximal activity (V_max) of electron transport system complexes I (A) and II (B) in liver mitochondria from ground squirrels in torpor (n = 6) and interbout euthermia (IBE, n = 6) after incubation with λ-protein phosphatase (LPP). *P ≤ 0.05 between torpor and IBE within a treatment; #P ≤ 0.05 within torpor or IBE.

Fig. 5.

Maximal activity of electron transport system complexes I (A) and II (B) in liver mitochondria from ground squirrels in torpor (n = 6) and interbout euthermia (IBE, n = 6) after incubation with protein kinase A (PKA). *P ≤ 0.05 between torpor and IBE within a treatment.