NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix

Abstract

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression

“…Immunoprecipitation of NME6-MycFlag coupled with mass spectrometry identified the putative mitochondrial ribosome assembly factor, RCC1L, to be the only high confidence interaction partner of NME6 (Fig. S4A), in line with previous proximity labelling and immunoprecipitation assays 41,52,53 . However, unlike cells depleted of RCC1L 54 , we did not observe any disruption of mitoribosome proteins in cells lacking NME6 (Fig.…”

“…1F) 37 . Three NME family members are reported to reside at mitochondria in different subcompartments; NME3 is located at the mitochondrial surface 38 , NME4 has been reported to be exposed to both the intermembrane space and matrix 39,40 and NME6 is imported to the mitochondrial matrix 41 . Since the reduction of mtDNA upon transfection with NME4 and NME6 sgRNA was subtle (Fig 1E ), we generated stable monoclonal knockout cells by plasmid expression of NME4 or NME6 sgRNA and determined mtDNA levels by realtime quantitative PCR (qPCR).…”

“…S1B, C). NME6 is ubiquitously expressed in humans and catalyses phosphotransfer through a conserved histidine residue within an NDPK consensus motif at position 137 (H137) 42 and kinase independent functions of NME6 have also been proposed 41,43 . Importantly, mtDNA levels were maintained in NME6 knockout cells expressing WT NME6-MycFlag but not in cells expressing kinase inactive mutant NME6 (NME6 H137N -MycFlag) (Fig.…”

The Complex Functions of the NME Family—A Matter of Location and Molecular Activity

“…In both compartments, NME4 is mainly bound to the inner mitochondrial membrane, interacting with cardiolipin, the mitochondrial signature phospholipid [ 11 ]. Most recent data reveal that NME6, the only ubiquitously expressed group II NME, also localizes to the mitochondrial inner membrane, mainly facing the matrix [ 12 ]. In contrast to NME4, NME6 is monomeric and NDP kinase inactive.…”

“…For example, NME1 binding to the key metabolite Coenzyme A (CoA) was reported, which is competitive in respect to nucleotides, and can lead to an inhibitory covalent CoA-modification of NME1 [ 26 ]. Most recently, mitochondrial NME6 was mainly localized in complexes with the regulator of chromatin condensation 1-like protein (RCC1L, also called WBSCR16) [ 12 ] that is involved in mitoribosome biogenesis and thus mitochondrial translation. RCC1L is part of a large GTP-binding protein family, but it is unknown whether it can function as a GDP/GTP exchange factor, and which would be the role of the kinase-inactive NME6 within a likely multi-protein regulatory complex.…”