To circumvent the long exposure times often associated with detection of radioactive fatty acid incorporated into proteins, we developed a two-step chemical method to detect palmitoylated proteins (). In the first step, a bio-orthogonal isosteric azido-palmitate analog (azido-tetradecanoate) is converted into its CoA-derivative by fatty acyl-CoA synthetase and incubated with mitochondrial proteins. In the second step, the azido-palmitoylated protein is reacted with a variety of tagged triaryl-phosphines (e.g., Myc, biotin, or fluorescein) for detection. The detection achieved with the various triarylphosphine tags was not only rapid and highly sensitive but also highly specific.
Our alternative methodology reduced the exposure times from days or months with autoradiographic/fluorographic methods, using [125
I]iodopalmitate or [3
H]palmitate as label, to seconds, with ECL or fluorescence imaging. Recently, Hang et al.
) also demonstrated that a variety of azido-fatty acid analogues can be used as chemical probes to monitor protein fatty acylation using the Staudinger ligation, therefore establishing a proof-of-principle for such a methodology. Of relevance to our study, they also showed that 14-azidododecanoate (azido-palmitate herein) is preferentially incorporated into proteins via
thioester bonds. In the present study, we established the proof of principle with greater detail and versatility and exploited this new technique to demonstrate the existence of several palmitoylated proteins in rat liver mitochondria. Furthermore, using anio-exchange chromatography, in vitro
labeling, separation by SDS-PAGE, and mass spectrometry, we identified 21 of these palmitoylated proteins in a compartment not yet recognized for its content in palmitoylated proteins. Primary hepatocyte labeling with radioactive palmitate () and labeling of mitochondria in vitro
(-) demonstrate that mitochondrial palmitoylated proteins are numerous and attest to the importance of investigating mitochondrial protein palmitoylation further. Of the bands identified, 19 of 21 contained peptides with masses that corresponded to only one protein, thus illustrating the efficiency of our strategy to identify these palmitoylated proteins.
The fact that two previously confirmed mitochondrial palmitoylated proteins (carbamoyl phosphate synthetase 1 and methylmalonyl semialdehyde dehydrogenase) were identified in this screen further validates our methodology (15
). Utilizing a recombinant HMGCS-His6
, we further compared the labeling properties and efficiencies of traditional radiolabeling to azido-palmitate labeling in vitro
. The newly identified palmitoylated protein, mitochondrial HMGCS-His6
, incorporated the traditional radiolabeled fatty acid analog. It also incorporated the azido-palmitate followed by tagged phosphine detection on a cysteine residue via
a thioester bond ( and ) at the same sites since azido-palmitate incorporation could be competed out by palmitoyl-CoA preincubation ().
HMGCS is the rate-limiting enzyme in ketogenesis, the process of converting acetyl-CoA, derived mainly from β-oxidation of fatty acids, into ketone bodies. Interestingly, we and others have demonstrated that palmitoyl-CoA inhibits HMG-CoA synthase (data not shown; also, see ref. 40
). Because the concentrations of LCFA-CoAs are increased in the liver mitochondria when ketone production is necessary, e.g.
, during fasting or starvation (42
), we do not expect that inhibition of the enzyme by palmitoylation is the main function for this type of modification. Hence, further investigation should focus on possible roles for its palmitoylation.
A large group of the identified palmitoylated proteins (9 of 21) are dehydrogenases (Supplemental Tables 1 and 3
). We believe, as proposed earlier (22
), that the internal NAD(H) binding site of these dehydrogenases could help facilitate acylation of the enzymes through the binding of the ADP-ribose moiety of acyl-CoA, which brings the thioester bond into close proximity to a nucleophilic cysteine residue. The previous demonstration of the palmitoylation of bovine liver glutamate dehydrogenase (EC 188.8.131.52) (20
) and methylmalonyl semialdehyde dehydrogenase (20
) also suggests that a significant subset of mitochondrial dehydrogenases could be potential substrates for palmitoylation. This is also corroborated by the identification of a palmitoylation site in the cytosolic glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (22
Palmitoylation of these dehydrogenases and of the other identified mitochondrial enzymes appeared to require only the coenzyme A form of long-chain fatty acid and purified enzyme, thereby suggesting that the reaction occurs in a spontaneous fashion. Further to this point, fatty acylation of the recombinant HMGCS-His6 purified from bacteria required only the presence of LCFA-CoA analogues, which thus ruled out that contamination by an “acylating mitochondrial transferase” is required for the acylation of mitochondrial proteins in vitro.
The alpha and beta subunits of the electron-transferring flavoprotein (ETF) as well as 3 of their electron donating dehydrogenases—sarcosine dehydrogenase, isovaleryl-CoA dehydrogenase, and dimethylglycine dehydrogenase (44
)—were identified as palmitoylated. This finding suggests that palmitoylation is a possible requirement for their membrane localization and subcompartmentalization in close vicinity to one another and to the ETF:ubiquinone oxidoreductase (ETF-QO) and complex III of the electron transport chain, their downstream electron acceptors (46
The ETF also serves as the electron acceptor for the various β-oxidation acyl-CoA dehydrogenases (50
). We also show that the β-oxidation enzymes 3-ketoacyl-CoA thiolase (EC 184.108.40.206), hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase (EC 220.127.116.11), and long-chain specific acyl-CoA dehydrogenase appear to be palmitoylated on a cysteine residue. Of note, short-chain specific acyl-CoA dehydrogenase (EC 18.104.22.168) was identified in a previous screen using [125
I]-iodo-palmitoyl-CoA as a label (data not shown). One could argue that these enzymes may form covalent acyl-enzyme intermediates during catalysis, like we have seen with fatty acyl-CoA synthetase ( and -). Arguing in favor of this possibility is the fact that 3-ketoacyl-CoA thiolase is known to form a thioester intermediate (55
), but arguing against it are the facts that enoyl-CoA hydratase, hydroxyacyl-CoA hydratase, and the acyl-CoA dehydrogenases are not reported to use cysteine residues during catalysis nor to form thioester intermediates (54
). We believe that palmitoylation of these enzymes might provide a membrane anchor and offer a plausible mechanistic explanation for the known localization of these enzymes and of others (59
) to the inner mitochondrial membrane.
Also detected as putative palmitoylated proteins in this screen were the key enzymes of the malate aspartate shuttle, the malate dehydrogenase (EC 22.214.171.124), and the aspartate aminotransferase (EC 126.96.36.199). In vitro
binding experiments have revealed that the addition of palmitoyl-CoA enhances the binding of glutamate dehydrogenase to aspartate aminotransferase or malate dehydrogenase (63
) and that ternary complexes of all three enzymes could be formed (65
). In contrast, addition of glutamate dehydrogenase could prevent the binding of citrate synthase to malate dehydrogenase in the presence of palmitoyl-CoA (63
). From these observations and since glutamate dehydrogenase can be palmitoylated in vitro
), we can speculate that selective palmitoylation of these enzymes may function as a general mechanism to promote or stabilize protein–protein interactions and possibly perform a regulatory role in the directional flow of the malate-aspartate shuttle. During conditions of high palmitoyl-CoA as seen in obesity, disregulation of the malate-aspartate shuttle could contribute to the increased gluconeogenesis commonly found in type II diabetes (23
We also identified alanine-glyoxylate aminotransferase 2 (EC 188.8.131.52 and EC 184.108.40.206) as a putative palmitoylated protein. This enzyme catalyzes the reversible transamination between alanine and pyruvate, utilizing either glycine and glyoxylate or glutamate and α-ketoglutarate as substrates (EC 220.127.116.11) or methylmalonate semialdehyde and 3-amino-isobutyrate as substrates (EC 18.104.22.168). Note that alanine glyoxylate aminotransferase competes with methylmalonyl semialdehyde dehydrogenase for a common substrate, methylmalonate semialdehyde, a product of valine catabolism, and that methylmalonyl semialdehyde dehydrogenase is inhibited by fatty acylation (20
). It is unknown how palmitoylation affects alanine glyoxylate aminotransferase, but under conditions of high fatty acyl-CoA the inhibition of methylmalonyl semialdehyde dehydrogenase would free up the methylmalonate semialdehyde to be used for the conversion of alanine to pyruvate by alanine glyoxylate aminotransferase. Should palmitoylation have a positive affect on the activity of alanine glyoxylate aminotransferase, then increased levels of LCFA-CoA in mitochondria could result in the disregulation of pyruvate production favoring gluconeogenesis over glycolysis.
Other than providing possible membrane tethering, we also found proteins for which we could not offer simple alternative explanations for possible palmitoylation-related functions. These are alpha-methylacyl-CoA racemase (EC 22.214.171.124), sulfite oxidase (EC 126.96.36.199), and—surprisingly—the heat-shock protein 75 kDa chaperone. In addition to the above, we identified the hypothetical protein LOC365699, with homology to sugar, NAD, and sphingosine kinases, as potentially palmitoylated.
The chemical detection of palmitoylated protein using a bio-orthogonal azido-palmitate analog has multiple advantages. Since azido-fatty acids are abiotic and nontoxic to cells or animals and are readily converted into acyl-CoAs by fatty acyl-CoA synthetase (30
), they could eventually be used to monitor protein palmitoylation in vivo
and to perform activity based protein profiling studies (66
). Furthermore, the potential use of a phosphine-biotin tag could allow for wider scale proteomics analyses using avidin-based technology. On a smaller scale herein, we demonstrated the existence of many palmitoylated proteins in rat liver mitochondria, identified several of these proteins, and postulated that mitochondrial protein palmitoylation could represent an active regulator of intermediary metabolism.