Elsevier

Cellular Signalling

Volume 20, Issue 5, May 2008, Pages 989-997
Cellular Signalling

cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I

https://doi.org/10.1016/j.cellsig.2008.01.017Get rights and content

Abstract

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.

Introduction

All the subunits of mitochondrial oxidative phosphorylation complexes, except 13 encoded by mitochondrial DNA, are encoded by nuclear genes, synthesized in the cytosol and imported into mitochondria. Complex I (NADH–ubiquinone oxidoreductase, E.C 1.6.5.3) of the mammalian mitochondrial respiratory chain consists of 45 subunits [1], [2]. Fourteen are conserved from prokaryotes to humans, these contain all the known redox centers of the complex [1], [2], [3], [4]. In mammals seven conserved subunits are encoded by the mitochondrial DNA [5], all the others by nuclear genes [6]. The functional role of the supernumerary subunits is not yet understood. Some of them appear to be essential for the assembly of the enzyme [7], [8], [9], for others analogy with known proteins of other systems has been found [10], [11].

Biogenesis of oxidative phosphorylation complexes depends on the concerted expression of mitochondrial and nuclear genes coding for their subunits [12]. The subunits encoded by nuclear genes have mitochondrial targeting information on specific sequences, either internal to the mature protein or as a cleavable N-terminal leader sequence [13]. Those with NH2-terminal leader sequences are imported by the mitochondrial outer membrane (TOM)-inner membrane (TIM) system [14]. Once the precursor proteins have reached the inner-mitochondrial compartment, the leader sequences are cleaved off by mitochondrial peptidases, thus generating the mature forms that are finally assembled in the functional enzyme [15], [16].

In murine [17], [18], [19] and human cell cultures in vivo[7], [20] cAMP has been found to promote the NADH–ubiquinone oxidoreductase activity of complex I and to prevent mitochondrial production of reactive oxygen species [21], [22]. These effects appear to be associated with cAMP-dependent phosphorylation by PKA of an 18-kDa subunit of complex I, identified, in these conditions, as that encoded by the nuclear NDUFS4 gene [[7], [17], [18], [19]]. Pathological mutations in human NDUFS4 exons [[7], [23], [24], [25]] have been found to result in disappearance of the protein encoded by the gene, failure of a final step of complex assembly [7], [8], [26], suppression of the NADH–ubiquinone oxidoreductase activity and its stimulation by cAMP [7], [20].

It has been reported that phosphorylation by PKA promotes the import in mitochondria of proteins like cytochrome P4502B1[27], CYP2E1 [28] and glutathione S-transferase (GSTA4-4) [29].

Work is presented here on the effect of phosphorylation by PKA of the NDUFS4 protein, synthesized in vitro or in vivo in HeLa cells, on its import and accumulation as mature form in mitochondria. The human NDUFS4, used in this investigation, has two conserved serine phosphorylation consensus sites (EMBL Data Bank), one in the NH2-terminal leader sequence, the other in the carboxy-terminal tail (Fig. 1). The impact of PKA and alkaline phosphatase (AP) on the import and accumulation as mature form in mitochondria of another human, complex I subunit (ESSS), encoded by the nuclear gene NDUFB11, which has only one serine phosphorylation consensus site, at the junction between the leader and the mature sequence, has also been investigated. The bovine ESSS has also a consensus serine phosphorylation site in the mature sequence [30], which, however, is absent in the human subunit (Fig. 1, EMBL Data Bank). The present results show that the accumulation in mitochondria of the mature NDUFS4 protein is specifically promoted by PKA-catalyzed phosphorylation of the serine at the C-terminal CPS and depressed by AP. PKA and AP had, on the other hand, no effect on the import/maturation of the ESSS subunit. The PKA promoting effect on the mitochondrial accumulation of NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol, through interaction of the phosphorylated form with the cytosolic Hsp70.

Section snippets

Materials

3-isobutyl-1-methylxanthine (IBMX), PKI and protein A-sepharose from Sigma, USA; cPKA (catalytic subunit cAMP-dependent protein kinase), Promega, USA; RII subunit (alpha regulatory subunit of PKA), Biaffin, Germany; alkaline phosphatase, Roche, Germany; [35S]Methionine (1000 Ci/mmol), Amersham Biosciences, United Kingdom; [γ-32P]ATP (3000 Ci/mmol), Perkin Elmer, USA.

Wild type and mutant cDNA constructs and in vitro translation of NDUFS4 and ESSS proteins

Full-length human NDUFS4 and ESSS cDNAs were generated by reverse transcriptase-PCR, using RNA extracted from primary fibroblast

Phosphorylation of in vitro synthesized NDUFS4 protein promotes accumulation of the mature form in mitochondria

The precursor form of the NDUFS4 protein produced in vitro in the RRL system, was detected by autoradiography, when obtained in the presence of [35S]Methionine, and recognized by antibodies raised against its N-terminal or phosphorylated C-terminal segments (Fig. 2). In the presence of added cPKA the protein was phosphorylated by [γ-32P]ATP (Fig. 2A). Immunoblot assays (Fig. 2A and B) showed that phosphorylation of the precursor NDUFS4 protein, promoted by PKA or depressed by AP, had no effect

Discussion

Whilst the mitochondrial encoded subunits of complex I are directly synthesized in the inner-mitochondrial compartment, the nuclear encoded subunits have to be imported in the required amount into mitochondria after they are synthesized in the cytosol. The NDUFS4 (AQDQ) and NDUFB11 (ESSS) subunits of complex I have canonical, positively charged leader sequences (Fig. 1), characteristic for the Δψ and ATP-dependent TOM/TIM mitochondrial import process [34] (Fig. 3, Fig. 4). The present study

Acknowledgements

This work was supported by: National Project on “Molecular Mechanisms, Physiology and Pathology of Membrane Bioenergetics System”, 2005-Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Italy and Research Foundation Cassa di Risparmio di Puglia.

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