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. 2012 May;23(9):1618-27.
doi: 10.1091/mbc.E11-11-0933. Epub 2012 Mar 14.

Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import

Sanjana Rao  1 , Oliver Schmidt, Angelika B Harbauer, Birgit Schönfisch, Bernard Guiard, Nikolaus Pfanner, Chris Meisinger
Affiliations

Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import

Sanjana Rao et al. Mol Biol Cell. 2012 May.

Abstract

The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22, and Tom70 and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the nonphosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but it also inhibits the biogenesis of the channel protein Tom40.

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Figures

FIGURE 1:
FIGURE 1:
Phosphorylation of Tom40 by yeast PKA. (A) Yeast cells from rho+ wild-type (WT), rho− wild type, and bcy1Δ were grown on sucrose-containing medium at 30°C. Protein extracts were prepared by postalkaline lysis and analyzed by SDS–PAGE and Western blotting. Alo1, d-arabinono-1,4-lactone oxidase; Pgk1, 3-phosphoglycerate kinase. (B) Yeast PKA (Bcy1-Tpk) was purified by affinity chromatography using a Bcy1ProtA yeast strain as described in Materials and Methods. Total (yeast lysate) and TEV eluate were analyzed by SDS–PAGE and Western blotting. Total, 10%; eluate, 100%. Cka1, Cka2, catalytic subunits of CK2; Ssa1, cytosolic member of heat shock protein 70 family. (C) Kemptide, the cytosolic domain of Tom70, and recombinant Tom40 (WT and S54E mutant form) were incubated with purified yeast PKA (Bcy1-Tpk) and [γ-33P]ATP in the presence of 8Br-cAMP as indicated. The samples were analyzed by SDS–PAGE, digital autoradiography, and staining with Coomassie brilliant blue R-250. Arrowhead, bovine serum albumin. (D) Recombinant Tom40WT and Tom40S54E were incubated with affinity-purified yeast Tpk1 as indicated and [γ-33P]ATP. The samples were analyzed as described for C.
FIGURE 2:
FIGURE 2:
PKA phosphorylates the precursor of Tom40 at serine 54. (A) Recombinant Tom40WT or Tom40S54A was incubated with purified PKA (New England BioLabs). The samples were lysed in Laemmli buffer and analyzed by Phos-tag SDS–PAGE and Western blotting using antiserum directed against Tom40. (B) Mitochondria were isolated from WT and Tom40S54A yeast strains and incubated with purified yeast PKA (Bcy1-Tpk) and 8Br-cAMP for the indicated periods. Where indicated, the samples were subsequently incubated with alkaline phosphatase (AP). The samples were analyzed as described for A. Similarly, mammalian PKA did not phosphorylate Ser-54 of Tom40 in intact mitochondria. (C) 35S-Labeled Tom40WT and Tom40S54A were synthesized in reticulocyte lysate in the presence or absence of PKA as indicated. Sample 6 was subsequently treated with AP. The samples were analyzed by Phos-tag SDS–PAGE and digital autoradiography. Arrowhead, nonspecific band; Tom40P, phosphorylated form of Tom40.
FIGURE 3:
FIGURE 3:
Replacement of serine 54 by glutamate impairs import of Tom40 into mitochondria. 35S-Labeled Tom40S54E, Tom40WT, and Tom40S54A precursors were incubated with isolated yeast wild-type mitochondria at 25°C for the indicated periods. The samples were split in half, and the mitochondria were reisolated. (A) One half was lysed in Laemmli buffer and analyzed by SDS–PAGE and digital autoradiography. (B) The other half was resuspended in Na2CO3, pH 11.5, and incubated for 30 min on ice. Membrane sheets were pelleted by centrifugation at 100,000 × g and analyzed by SDS–PAGE and digital autoradiography.
FIGURE 4:
FIGURE 4:
Phosphorylation of Tom40 by PKA impairs formation of the SAM import intermediate. (A) 35S-Labeled precursor of Tom40 was synthesized in the presence or absence of PKA (New England BioLabs) as indicated and incubated with isolated yeast wild-type mitochondria for the indicated periods at 25°C. The mitochondria were solubilized in digitonin-containing buffer and analyzed by blue native electrophoresis and digital autoradiography. (B) 35S-Labeled precursors of Tom6 and Tom7 were imported into isolated mitochondria in the presence or absence of PKA. The mitochondria were analyzed as described for A. (C) [35S]Tom40WT and [35S]Tom40S54A precursors were synthesized in the presence or absence of PKA and incubated with isolated mitochondria for 5 min at 25°C. The mitochondria were analyzed as described for A.
FIGURE 5:
FIGURE 5:
Mitochondria import the nonphosphorylated precursor of Tom40, whereas phosphorylated Tom40 remains on the mitochondrial surface. (A) [35S]Tom40 was synthesized in the presence of PKA (New England BioLabs) and incubated with isolated wild-type mitochondria for the indicated periods at 25°C. The mitochondria were treated with proteinase K (Prot. K; Stojanovski et al., 2007) where indicated and analyzed by Phos-tag SDS–PAGE and digital autoradiography. (B) [35S]Tom40 was synthesized in the presence or absence of PKA and imported into mitochondria that had been pretreated with trypsin (Ryan et al., 2001) as indicated. The mitochondria were analyzed by Phos-tag SDS–PAGE. (C) [35S]Tom40 was imported into mitochondria, which were isolated from tom22Δ yeast or the corresponding wild-type (WT) strain, in the presence or absence of PKA. The mitochondria were treated with proteinase K where indicated and analyzed by Phos-tag SDS–PAGE. (D) [35S]Tom40 was imported into mitochondria, which were isolated from tom20Δ and wild-type yeast, in the presence or absence of PKA. The mitochondria were analyzed by Phos-tag SDS–PAGE. (E) [35S]Tom40 was imported into mitochondria, which were isolated from tom70Δ and wild-type yeast, in the presence of PKA. The mitochondria were treated with proteinase K where indicated and analyzed by Phos-tag SDS–PAGE. (F) Mitochondria were preincubated in isotonic or hypotonic (swelling) buffer for 30 min on ice (Stojanovski et al., 2007). The mitochondria/mitoplasts were reisolated and incubated with [35S]Tom40 in the presence or absence of PKA and analyzed by Phos-tag SDS–PAGE. (G) [35S]Tom40 was imported into mitochondria, which were isolated from tim10-2 yeast or the corresponding wild-type strain, in the presence or absence of PKA. The mitochondria were analyzed by Phos-tag SDS–PAGE.
FIGURE 6:
FIGURE 6:
Inhibition of Tom40 import by PKA is not connected to the phosphorylation of Tom70 by PKA. 35S-Labeled precursor of Tom40 was imported into mitochondria, which were isolated from Tom70S174E, Tom70WT, or Tom70S174A yeast strains, at 25°C in the presence or absence of PKA (New England BioLabs) as indicated. The mitochondria were solubilized in digitonin-containing buffer and analyzed by blue native electrophoresis and digital autoradiography.
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References

    1. Abe Y, Shodai T, Muto T, Mihara K, Torii H, Nishikawa S, Endo T, Kohda D. Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell. 2000;100:551–560. - PubMed
    1. Ahting U, Thieffry M, Engelhardt H, Hegerl R, Neupert W, Nussberger S. Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria. J Cell Biol. 2001;153:1151–1160. - PMC - PubMed
    1. Albuquerque CP, Smolka MB, Payne SH, Bafna V, Eng J, Zhou H. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics. 2008;7:1389–1396. - PMC - PubMed
    1. Anandatheerthavarada HK, Biswas G, Mullick J, Sepuri NB, Otvos L, Pain D, Avadhani NG. Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128. EMBO J. 1999;18:5494–5504. - PMC - PubMed
    1. Becker L, Bannwarth M, Meisinger C, Hill K, Krimmer T, Casadio R, Truscott KN, Schulz GE, Pfanner N, Wagner R. Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol. 2005;353:1011–1020. - PubMed

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