bims-tricox 2023-08-20 papers

Issue of 2023‒08‒20
four papers selected by
Yash Verma, University of Zurich

Life Sci Alliance. 2023 Nov;pii: e202302004. [Epub ahead of print]6(11):

A Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of T. brucei.

Markus Gerber, Ida Suppanz, Silke Oeljeklaus, Moritz Niemann, Sandro Käser, Bettina Warscheid, André Schneider, Caroline E Dewar.

The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

DOI: https://doi.org/10.26508/lsa.202302004

Proc Natl Acad Sci U S A. 2023 08 22. 120(34): e2301447120

Two conformations of the Tom20 preprotein receptor in the TOM holo complex.

Pamela Ornelas, Thomas Bausewein, Janosch Martin, Nina Morgner, Stephan Nussberger, Werner Kühlbrandt.

The TOM complex is the main entry point for precursor proteins (preproteins) into mitochondria. Preproteins containing targeting sequences are recognized by the TOM complex and imported into mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle electron cryomicroscopy at 3.3 Å resolution, showing its interaction with a bound preprotein at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6 to 7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits, and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

Keywords: TOM complex; Tom20; cryoEM; translocation

DOI: https://doi.org/10.1073/pnas.2301447120

PLoS Genet. 2023 Aug 16. 19(8): e1010848

Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import.

Salomé Nashed, Houssam El Barbry, Médine Benchouaia, Angélie Dijoux-Maréchal, Thierry Delaveau, Nadia Ruiz-Gutierrez, Lucie Gaulier, Déborah Tribouillard-Tanvier, Guillaume Chevreux, Stéphane Le Crom, Benoit Palancade, Frédéric Devaux, Elodie Laine, Mathilde Garcia.

N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.

DOI: https://doi.org/10.1371/journal.pgen.1010848

bioRxiv. 2023 Aug 04. pii: 2023.08.04.552005. [Epub ahead of print]

Identification of factors that prevent ribosome stalling during early elongation.

Hye-Rim Hong, Cassidy R Prince, Letian Wu, Heather A Feaga.

Protein synthesis is catalyzed by the ribosome and a host of highly conserved elongation factors. Most elongation factors that are conserved in all domains of life are essential, such as EF-Tu (e/aEF1A) and EF-G (e/aEF2). In contrast, the universally conserved elongation factor P (EF-P/eIF5A) is essential in eukaryotes but is dispensable in most bacteria. EF-P prevents ribosome stalling at difficult-to translate sequences, especially polyprolines. Since efp deletion phenotypes range from modest to lethal in different bacterial species, we hypothesized that some bacteria encode an uncharacterized elongation factor with compensatory functions. To identify this factor, we used Tn-seq to screen for genes that are essential in the absence of EF-P in Bacillus subtilis . This screen identified YfmR, a member of the ABCF family of ATPases, as a translation factor that is essential when efp is deleted. We find that depleting YfmR from Δ efp cells decreases actively translating ribosomes and increases free ribosomal subunits bound to initiator tRNA, suggesting that ribosomes stall in early elongation. We also find that deleting efp from the spore-forming pathogen Bacillus anthracis causes a severe survival defect in liquid culture and in the presence of macrophages. B. anthracis Δefp also does not produce detectable spores. We find that heterologous expression of B. subtilis YfmR in B. anthracis Δ efp cells partially rescues the severe growth and sporulation defects of this mutant. Our results therefore identify YfmR as an important translation factor that becomes essential in the absence of EF-P, and suggest that the essential function of YfmR and EF-P is to assist translation during early elongation.Significance: Translation is one of the most ancient and energetically demanding process that occurs in the cell. Ribosomes constitute more than 60% of cellular mass in actively growing cells, and ribosomes are a major target of antimicrobials and chemotherapeutics. Here, we report evidence that both EF-P and the uncharacterized protein YfmR are important for early translation elongation in bacteria, and that this activity is essential. YfmR is a member of the ABCF family of ATPases whose role in translation is only beginning to be understood. Given the broad distribution of ABCFs from bacteria to fungi, we expect our results to have implications for understanding the crucial first steps of translation elongation in diverse organisms.

DOI: https://doi.org/10.1101/2023.08.04.552005