Presequences of non-imported mitochondrial proteins serve as quality control elements in the cytosol

Djp1 is a multifunctional Hsp40 cochaperone for mitochondrial phospholipid metabolism

Mitochondria are cellular energy hubs best known for ATP production via oxidative phosphorylation; however, they also serve as biosynthetic centers for phospholipids. Mitochondrial phospholipids are critical for various cellular processes, and their loss underlies myriad mitochondrial diseases. The critical enzymes underlying these biosynthetic cascades are encoded in the nucleus, translated in the cytosol, and imported into mitochondria. Understanding of mechanisms and factors that ensure precise targeting of proteins to mitochondria has been long overlooked but remains critical. Recently, the J-protein/Hsp40 cochaperone Djp1 has emerged as a key player in mitochondrial protein targeting by promoting the transfer of precursors from the endoplasmic reticulum (ER) surface to mitochondria in a pathway termed ER-SURF. Molecular details regarding how Djp1 recognizes clients and more broadly supports mitochondrial function remain unknown. Using biochemical approaches, proteomics, and thin layer chromatography, we demonstrate that Djp1 is a regulator of Phosphatidylserine decarboxylase 1 (Psd1), an inner mitochondrial membrane resident responsible for mitochondrial phosphatidylethanolamine (PE) production. This regulation of Psd1 biogenesis is dependent on its mitochondrial targeting signal and is specific to Djp1 compared to other members of the Hsp40 family or ER targeting factors. Intriguingly, the combined loss of Djp1 and Psd1 results in a synthetic sick phenotype that unexpectedly reflects a role(s) for Djp1 in proper mitochondrial phospholipid metabolism independent of Psd1. Taken together, these findings expand our understanding of Djp1-dependent mitochondrial protein regulation and unveil Djp1 as important for mitochondrial phospholipid metabolism by multiple mechanisms.

Combinatorial sequence elements fine-tune mitochondrial protein import to facilitate dual localization

It is found that PPTC7 has a moderately ‘weak’ presequence, but this feature is insufficient to promote dual targeting of a generic cargo protein, and sequence-specific features can work combinatorially to impart dual-localization capacity to mitochondrial proteins, enabling functions across cellular compartments.

Selective quality control of mistargeted mitochondrial proteins at the endoplasmic reticulum

In eukaryotic cells, the function of each organelle depends on its unique protein composition. Protein targeting errors threaten organelle identity and function, yet how mistargeting errors are detected and resolved remains poorly understood. Here, we show that mitochondrial import stress drives widespread rerouting of mitochondrial proteins to the endoplasmic reticulum (ER), with strong enrichment for hydrophobic oxidative phosphorylation (OXPHOS) components. Using proximity proteomics, a split-fluorescence reporter system, and genome-wide CRISPR screening, we find that mistargeted proteins partition into distinct classes with divergent fates, ranging from stable ER residence to rapid degradation by ER-associated degradation (ERAD). The clearance of these mislocalized proteins involves partially redundant ERAD branches, with the ubiquitin ligase MARCHF6 playing a central role. Together, these findings establish the ER as a key organelle for handling mistargeted mitochondrial proteins and reveal how ER quality control maintains proteostasis during mitochondrial dysfunction.

A p32 family RNA editing factor acts in mitochondrial ribosome biogenesis

Biogenesis of mitochondrial ribosomes (mitoribosomes) in the unicellular parasite Trypanosoma brucei requires an exceptionally large toolkit of assembly factors, identified in stable precursors of large and small mitoribosomal subunits (mtLSU and mtSSU) by cryoEM. Here, using genetic modifications and proteomic characterization of the immunoprecipitated assemblosome, the earliest characterized mtSSU precursor, we determined that a cap of its distinctive protrusion of hitherto unknown composition consists of a p22 homotrimer. This protein was previously implicated in the uridine-insertion editing of the cytochrome c oxidase subunit II transcript. Our functional analysis confirmed this role but revealed that its ablation also causes a loss of mtSSU and a systemic reduction in mitochondrial translation, phenocopying the depletion of established mitoribosomal assembly factors. Consequently, the oxidative phosphorylation system and mitochondrial function are compromised. The p22 protein belongs to the p32 family. We showed that five of its six trypanosomal members are involved in mtSSU biogenesis. Notably, p32 proteins are associated with mitoribosomes in two other distant eukaryotic lineages. A eukaryote-wide mapping of p32 proteins documented that their presence correlates with the retention of mitochondrial genomes. Together, our findings redefine trypanosomal p22 as a dual-function coordinator of mitochondrial gene expression and reveal that the ancestral role of the p32 family is associated with mitochondrial translation.

Characterization of Mmp37p, a Characterization of Mmp37p, a Saccharomyces cerevisiae Mitochondrial Matrix Protein with a Role in Mitochondrial Protein Mitochondrial Matrix Protein with a Role in Mitochondrial Protein Import Import

Succinate and its carrier Sfc1 mediate metabolic control of mitochondrial protein import by the TIM23 translocase.

Tim23 is an essential component of the mitochondrial inner membrane translocase and Sfc1 is a carrier that exchanges succinate for fumarate across that membrane. Sfc1 and succinic acid availability regulate dual targeting of fumarase and aconitase by facilitating mitochondrial import of their newly synthesized precursors, as shown by pulse-chase experiments. Here, we show that Sfc1 associates with Tim23 in vivo, and succinate modulates this association, which in turn affects mitochondrial protein import. Physical interaction between Tim23 and Sfc1 was proven by co-immunoprecipitation, bimolecular fluorescence complementation (BiFC) and biotin-based proximity labeling (TurboID). Proximity labeling and structural modeling-informed mutagenesis allowed us to dissect the carrier activity of Sfc1 from its function as a TIM23 regulator. We performed Rosetta-MP docking of Sfc1 and Tim23 to envisage the interface. Thus, our findings show that metabolites can regulate mitochondrial import and adjust the segregation of key metabolic enzymes between the cytosol and mitochondria.