Biogenesis pathways of α-helical mitochondrial outer membrane proteins

Mitochondria harbor in their outer membrane (OM) proteins of different topologies. These proteins are encoded by the nuclear DNA, translated on cytosolic ribosomes and inserted into their target organelle by sophisticated protein import machineries. Recently, considerable insights have been accumulated on the insertion pathways of proteins into the mitochondrial OM. In contrast, little is known regarding the early cytosolic stages of their biogenesis. It is generally presumed that chaperones associate with these proteins following their synthesis in the cytosol, thereby keeping them in an import-competent conformation and preventing their aggregation and/or mis-folding and degradation. In this review, we outline the current knowledge about the biogenesis of different mitochondrial OM proteins with various topologies, and highlight the recent findings regarding their import pathways starting from early cytosolic events until their recognition on the mitochondrial surface that lead to their final insertion into the mitochondrial OM.

Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway

In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.

Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins

Mitochondrial β-barrel proteins are encoded in the nucleus, translated by cytosolic ribosomes, and then imported into the organelle. Recently, a detailed understanding of the intramitochondrial import pathway of β-barrel proteins was obtained. In contrast, it is still completely unclear how newly synthesized β-barrel proteins reach the mitochondrial surface in an import-competent conformation. In this study, we show that cytosolic Hsp70 chaperones and their Hsp40 cochaperones Ydj1 and Sis1 interact with newly synthesized β-barrel proteins. These interactions are highly relevant for proper biogenesis, as inhibiting the activity of the cytosolic Hsp70, preventing its docking to the mitochondrial receptor Tom70, or depleting both Ydj1 and Sis1 resulted in a significant reduction in the import of such substrates into mitochondria. Further experiments demonstrate that the interactions between β-barrel proteins and Hsp70 chaperones and their importance are conserved also in mammalian cells. Collectively, this study outlines a novel mechanism in the early events of the biogenesis of mitochondrial outer membrane β-barrel proteins.

Cytosolic ribosomes on the surface of mitochondria

By electron cryo-tomography and subtomogram averaging, translation-arrested ribosomes were used to depict the clustered organisation of the TOM complex on the surface of mitochondria, corroborating earlier reports of localized translation. Ribosomes were shown to interact specifically with the TOM complex and nascent chain binding was crucial for ribosome recruitment and stabilization. Ribosomes were bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport, and the importance of spatial organization for efficient mitochondrial protein import.

The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon

Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome–Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S–RQC and 80S–Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome–translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel.