Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMPdegrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.Mitochondria play central roles in cellular energy metabolism, as well as in the regulation of cell cycle progression, apoptosis, and aging processes (1, 2). Despite their importance, signaling into, from, and within mitochondria is still not well understood. Emerging signaling mechanisms in mitochondria and between the organelle and its environment include reversible protein deacetylation (3, 4), redox regulation and reactive oxygen species formation (5-7), and cyclic adenosine monophosphate (cAMP) signaling (8, 9).cAMP-dependent effects and proteins of cAMP signaling systems, such as cAMP-responsive element-binding protein (CREB), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), 3 have been described in mitochondria (10 -12). In addition to these effector proteins, a complete cAMP signaling microdomain requires enzymes for synthesis and degradation of the second messenger. Although an intramitochondrial cAMP source has been identified recently (8), there is no known cAMP-degrading enzyme in this organelle. Cyclic AMP is formed inside mitochondria by soluble adenylyl cyclase (sAC) (8), a member of Class III of the nucleotidyl cyclase family, which also comprises the G-protein-regulated transmembrane adenylyl cyclases (13). Unique from transmembrane adenylyl cyclases, sAC is activated by bicarbonate (14), and it appears to act as a metabolic sensor (15), whose mitochondrial form(s) seems to modulate PKA-mediated regulation of respiration (8) and apoptosis (16).The opponents of the cyclic nucleotide-forming cyclases are cyclic nucleotide monophosphate (cNMP)-degrading phosphodiesterases (PDEs). Mammalian cells contain a varying subset of members of the classical PDE family, which comprises 11 PDE gene families (PDE1-11) (17, 18) and non-generic PDEs such as the protein human Prune (19,20). The isoforms of the generic PDEs comprise homologous catalytic domains, fused to varying regulatory domains, making them sensitive to a variety of signals s...
The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.
Background The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported. Results We identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore. Conclusions The general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.
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