LONP1 Is Required for Maturation of a Subset of Mitochondrial Proteins, and Its Loss Elicits an Integrated Stress Response.

LONP1, an AAA+ mitochondrial protease, is implicated in protein quality control, but its precise role in this process remains poorly understood. In this study, we have investigated the role of human LONP1 in mitochondrial proteostasis and gene expression. Depletion of LONP1 resulted in partial loss of mitochondrial DNA (mtDNA) and a complete suppression of mitochondrial translation associated with impaired ribosome biogenesis. The levels of a distinct subset of mitochondrial matrix proteins (SSBP1, MTERFD3, FASTKD2, and CLPX) increased in the presence of a catalytically dead form of LONP1, suggesting that they are bona fide LONP1 substrates. Unexpectedly, the unprocessed forms of the same proteins also accumulated in an insoluble protein fraction. This subset of unprocessed matrix proteins (but not their mature forms) accumulated following depletion of the mitochondrial processing peptidase MPP, though all other MPP substrates investigated were processed normally. Prolonged depletion of LONP1 produced massive matrix protein aggregates, robustly activated the integrated stress response (ISR) pathway, and resulted in stabilization of PINK1, a mitophagy marker. These results demonstrate that LONP1 and MPPαß are together required for the maturation of a subset of LONP1 client proteins and that LONP1 activity is essential for the maintenance of mitochondrial proteostasis and gene expression.

Vms1p is a release factor for the ribosome-associated quality control complex.

Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.

A Mutation in the Flavin Adenine Dinucleotide-Dependent Oxidoreductase FOXRED1 Results in Cell-Type-Specific Assembly Defects in Oxidative Phosphorylation Complexes I and II.

Complex I (NADH ubiquinone oxidoreductase) is a large multisubunit enzyme that catalyzes the first step in oxidative phosphorylation (OXPHOS). In mammals, complex I biogenesis occurs in a stepwise manner, a process that requires the participation of several nucleus-encoded accessory proteins. The FAD-dependent oxidoreductase-containing domain 1 (FOXRED1) protein is a complex I assembly factor; however, its specific role in the assembly pathway remains poorly understood. We identified a homozygous missense mutation, c.1308 G→A (p.V421M) in FOXRED1 in a patient who presented with epilepsy and severe psychomotor retardation. A patient myoblast line showed a severe reduction in complex I, associated with the accumulation of subassemblies centered around ∼340 kDa, and a milder decrease in complex II, all of which were rescued by retroviral expression of wild-type FOXRED1. Two additional assembly factors, AIFM1 and ACAD9, coimmunoprecipitated with FOXRED1, and all were associated with a 370-kDa complex I subassembly that, together with a 315-kDa subassembly, forms the 550-kDa subcomplex. Loss of FOXRED1 function prevents efficient formation of this midassembly subcomplex. Although we could not identify subassemblies of complex II, our results establish that FOXRED1 function is both broader than expected, involving the assembly of two flavoprotein-containing OXPHOS complexes, and cell type specific.

The arginine methyltransferase NDUFAF7 is essential for complex I assembly and early vertebrate embryogenesis.

Complex I of the mitochondrial respiratory chain is a large multisubunit enzyme that assembles from nuclear and mtDNA-encoded components. Several complex I assembly factors have been identified, but their precise functions are not well understood. Here, we have investigated the function of one of these, NDUFAF7, a soluble matrix protein comprised of a DUF185 domain that harbors a methyltransferase motif. Knockdown of NDUFAF7 by siRNA in human fibroblasts produced a specific complex I assembly defect, as did morpholino-mediated knockdown of the zebrafish ortholog. Germline disruption of the murine ortholog was an early embryonic lethal. The complex I assembly defect was characterized by rapid, AFG3L2-dependent, turnover of newly synthesized ND1, the subunit that seeds the assembly pathway, and by decreased steady-state levels of several other structural subunits including NDUFS2, NDUFS1 and NDUFA9. Expression of an NDUFAF7 mutant (G124V), predicted to disrupt methyltransferase activity, impaired complex I assembly, suggesting an assembly factor or structural subunit as a substrate for methylation. To identify the NDUFAF7 substrate, we used an anti-ND1 antibody to immunoprecipitate complex I and its associated assembly factors, followed by mass spectrometry to detect posttranslational protein modifications. Analysis of an NDUFAF7 methyltransferase mutant showed a 10-fold reduction in an NDUFS2 peptide containing dimethylated Arg85, but a 5-fold reduction in three other NDUFS2 peptides. These results show that NDUFAF7 functions to methylate NDUFS2 after it assembles into a complex I, stabilizing an early intermediate in the assembly pathway, and that this function is essential for normal vertebrate development.

Early complex I assembly defects result in rapid turnover of the ND1 subunit.

Complex I (CI, NADH ubiquinone oxidoreductase), the largest complex of the respiratory chain, is composed of 45 structural subunits, 7 of which are encoded in mtDNA. At least 10 factors necessary for holoenzyme assembly have been identified; however, the specific roles of most of them are not well understood. We investigated the role of NDUFAF3, NDUFAF4, C8orf38 and C20orf7, four early assembly factors, in the translation of the mtDNA-encoded CI structural subunits. Transient, or stable, siRNA-mediated knock-down of any of these factors abrogated the assembly of CI, and resulted in a specific decrease in the labeling of the ND1 subunit in a pulse translation experiment, whereas knock-down of NDUFAF2, a late assembly factor, did not affect ND1 translation. Pulse-chase experiments in cells knocked down for NDUFAF3 showed that the half-life of ND1 in the chase was reduced 4-fold, fully accounting for the decrease in pulse labeling. Transient, short-term knock-down of the m-AAA protease AGF3L2 in cells that had been depleted of any of the early CI assembly factors completely rescued the ND1 labeling phenotype, confirming that it is not a synthesis defect, but rather results from rapid proteolysis of newly synthesized ND1. NDUFAF3 co-immunoprecipitated with NDUFAF4, and three matrix arm structural subunits (NDUFS2, NDUFA9, NDUFS3) that are found in a 400 kDa assembly intermediate containing ND1. These data suggest that the four early CI assembly factors have non-redundant functions in the assembly of a module that docks and stabilizes newly synthesized ND1, nucleating assembly of the holoenzyme.