To determine whether STAT3 regulates mitochondrial ATP generation via direct PPI with the complexes of oxidative phosphorylation, we first used two-dimensional gel electrophoresis to examine the stoichiometric relationship between STAT3 and Complexes I/II in purified heart mitochondria. Subsequently, we applied quantitative Western analyses and targeted mass spectrometry to obtain an absolute cellular concentration of STAT3 in total heart tissue.
In our experience, the complexes of oxidative phosphorylation are readily observed when heart mitochondrial protein extracts are resolved with two-dimensional gel techniques (5
). If STAT3 were stoichiometric with Complexes I/II, it would be detected at a molecular mass of 88 kDa and an isoelectric focusing point (pI) of 5.9. However, we could not detect any protein in this region of the gel (, A
). A two-dimensional Western analysis was then performed on heart mitochondria to eliminate the possibility that proteolysis or post-translational modifications altered the molecular weight or pI of STAT3 upon localization to the mitochondria. Again, STAT3 was not detected (, C
). To further establish the position of STAT3 in a complex mitochondrial protein mixture, porcine heart mitochondria were spiked with recombinant STAT3 at a 1:1 ratio with mitochondrial Complex I and visualized by two-dimensional DIGE (E
). Although the purified STAT3 was easily visualized at its expected molecular weight and pI (F
), again no STAT3 was detected in the unspiked mitochondrial sample (G
). Based on these experiments, we estimate that the ratio of Complex I to STAT3 must exceed several orders of magnitude.
FIGURE 1. Relationship of heart mitochondrial proteins and STAT3. A and B, representative two-dimensional gels of porcine (A) and murine (B) heart mitochondrial proteins, with an arrow indicating the expected position of STAT3. C and D, representative two-dimensional (more ...)
To confirm these findings, we searched mass spectrometry databases of mitochondrial proteins (MitoCarta (14
) and MitoP2 (15
)) and data from our laboratory in porcine (5
), rat (16
), and murine mitochondria but found no evidence of STAT3 localization to heart or liver mitochondria (). Collectively, these data are consistent with the notion that STAT3 levels are much lower than Complex I in heart mitochondria.
To determine the absolute cellular concentration of STAT3, quantitative Western blotting of total porcine heart proteins was performed, using recombinant STAT3 as a standard (A
). These experiments revealed that there are ~0.7 fmol of STAT3 per mg of porcine heart protein. To validate this finding, we utilized a targeted mass spectrometry approach against peptide IVELFR from STAT3 trypsin digestion (, B
), which yielded ~0.4 fmol of STAT3 per mg of porcine heart protein. Using 300 pg of total protein per cell as a conversion factor (12
), these methods establish the cellular concentration of STAT3 to be 73–123 molecules/cell (D
To quantitatively assess the relationship between STAT3 and Complexes I/II, we next determined the absolute concentration of the oxidative phosphorylation complexes in porcine heart mitochondria. Applying spectrophotometric techniques, we determined the concentration of Complex IV in porcine heart tissue to be 226 pmol/mg of protein, which is in good agreement with previous studies (8
). We next used previously determined ratios for the oxidative phosphorylation complexes, CI1
), and calculated the cellular concentrations of Complexes I/II to be 35 and 52 pmol/mg of protein, respectively. Assuming 300 pg of protein per cell, this translates to more than 6 million molecules of Complex I and 9 million molecules of Complex II per cell as compared with ~100 molecules of STAT3 per cell (D
). This result implies that the ratio of Complexes I/II to STAT3 is on the order of 105
, in agreement with our two-dimensional gel electrophoresis studies in .
Using these various methods to detect STAT3, we determined that its absolute concentration is on the order of 0.5 fmol/mg of protein or 100 molecules/cell. This low level of protein expression is consistent with other cellular transcription factors (17
). Indeed, most modeling efforts place transcription factors in the high picomolar to low micromolar range (20
). The small number of transcription factor molecules is likely due to the limited number of DNA targets responsible for controlling protein expression. Relative to Complexes I/II, our studies reveal that STAT3 is 105
-fold less abundant. This low concentration implies that there are not enough STAT3 molecules for every mitochondrion in the heart cell, let alone enough STAT3 molecules to bind to each molecule of Complexes I/II. Because only a vanishingly small fraction of Complexes I/II can be influenced by STAT3, it is highly unlikely that under normal conditions, protein-protein interaction between STAT3 and Complexes I/II could considerably alter oxidative phosphorylation. In fact, if the number of STAT3 molecules was increased to the level required for PPI with Complexes I/II (i.e.
from 100 molecules/cell to more than 1million molecules/cell), it is likely that STAT3 would saturate the binding sites on DNA and lead to defective protein expression.
Given the central role of the mitochondrion in cellular energy metabolism, redox regulation, apoptosis, heme and protein processing, and various biosynthetic and anabolic reaction pathways, it would be surprising if the elimination or overexpression of any general transcription factor did not affect mitochondrial programming and regulation. In the case of STAT3, the high ratio of Complexes I/II to STAT3 implies that PPI between these molecules would be highly ineffective in regulating oxidative phosphorylation. It is possible that STAT3 activates a small fraction of oxidative phosphorylation at the level of Complexes I/II via protein association. However, previous studies demonstrate that porcine, canine, and murine heart need to acutely regulate mitochondrial ATP production near Vmax in vivo
) and, therefore, require virtually complete activation of the oxidative phosphorylation complexes. This necessitates that a near equimolar concentration of STAT3 and Complexes I/II is present in cardiac mitochondria to generate sufficient ATP to support maximum cardiac performance. The current study revealed that the cellular ratio of Complexes I/II to STAT3 is not 1:1, but rather 105
, which implies that STAT3 is not modulating cardiac ATP generation via direct PPIs with the complexes of oxidative phosphorylation. We suggest that the effect of STAT3 on mitochondrial function, as demonstrated in previous studies (2
), may result instead from amplified indirect events such as protein expression regulation, alternate signaling pathways, or other undefined cooperative mechanisms that clearly warrant further investigation.