The molecular mechanisms of the mitoRQC pathway in the maintenance of mitochondrial homeostasis
Final Report Abstract
Eukaryotic cells evolved a number of different quality control mechanisms to deal with faulty proteins. One of the earliest acting ones is the ribosome-associated quality control (RQC). The project aimed at molecular understanding of the specialised RQC pathway acting on nuclear-encoded mitochondrial proteins, named mitoRQC. In the classical RQC pathway, Rqc2 recognizes obstructed 60S subunit with the associated peptidyl-tRNA. Ltn1, an E3 ubiquitin ligase, then ubiquitylates the faulty nascent chain marking it for degradation. Rqc2 also noncanonically extends the nascent chain by adding C-terminal alanine and threonine residues. These CAT tails serve as degrons but they also render proteins aggregation-prone. Mitochondria are particularly sensitive to CAT-tailed proteins – they aggregate in the mitochondrial matrix compromising mitochondrial function and ultimately resulting in cell death. Furthermore, due to the presence of N-terminal targeting signals, translocation of faulty nascent chains into mitochondria can be initiated before they are released from the ribosomes, making them partly or completely inaccessible to ubiquitylation by Ltn1. Mitochondria therefore depend on the protective function of the cytosolic protein Vms1 which releases faulty but not CAT-tailed proteins into mitochondria. Here, in collaboration with the group of Prof. Roland Beckmann, we solved the cryoEM structure of Vms1 bound to 60S subunit. Vms1 binds to 60S with its VLRF1, zinc finger and ankyrin domains. VLRF1 binds in and below A-site, interacting both with rRNA and peptidyl-tRNA. The Vms1 loop with the catalytic GSQ motif projects towards the CCA end of tRNA, remodelling the ribose-phosphate backbone and exposing it for nucleolytic cleavage. Unexpectedly, the structure revealed the presence of Arb1, an ABCF-type ATPase, in the E-site. Arb1 stabilizes the distorted conformation of tRNA and stimulates the Vms1-depedent tRNA cleavage. In the second line of research, we identified Pth2 as an additional factor involved in mitoRQC. This peptidyl-tRNA hydrolase in the outer membrane influences aggregation of CAT- tailed proteins without majorly affecting the CAT-tailing process itself. The hydrolase activity is essential during this process, yet it can be substituted by another peptidyl-tRNA hydrolase, upon proper localization. Pth2 appears to act through modulation of protein translocation and mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Analysis of other novel factors involved in mitoRQC show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.
Publications
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Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis. Nature, 570(7762), 538-542.
Su, Ting; Izawa, Toshiaki; Thoms, Matthias; Yamashita, Yui; Cheng, Jingdong; Berninghausen, Otto; Hartl, F. Ulrich; Inada, Toshifumi; Neupert, Walter & Beckmann, Roland
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How to get to the other side of the mitochondrial inner membrane – the protein import motor. Biological Chemistry, 401(6-7), 723-736.
Mokranjac, Dejana
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Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes. Life Science Alliance, 6(12), e202302122.
Genge, Marcel G.; Roy, Chowdhury Shalini; Dohnálek, Vít; Yunoki, Kaori; Hirashima, Takashi; Endo, Toshiya; Doležal, Pavel & Mokranjac, Dejana
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In vitro import of mitochondrial precursor proteins into yeast mitochondria. Methods in Enzymology, 347-363. Elsevier.
Badrie S.; Draken J.A. & Mokranjac D.
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Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins. Molecular Cell, 85(21), 4082-4092.e7.
Bertram, Nils; Izawa, Toshiaki; Thoma, Felix; Schwenkert, Serena; Duvezin-Caubet, Stéphane; Park, Sae-Hun; Wagener, Nikola; Devin, Anne; Osman, Christof; Neupert, Walter & Mokranjac, Dejana
