Premature arrest of protein synthesis within the open reading frame elicits a protective response that degrades the incomplete nascent chain. In this response, arrested 80S ribosomes are split into their large and small subunits, allowing assembly of the ribosome quality control complex (RQC), which targets nascent chains for degradation. How the cell recognizes arrested nascent chains among the vast pool of actively translating polypeptides is poorly understood. We systematically examined translation arrest and modification of nascent chains in Saccharomyces cerevisiae to characterize the steps that couple arrest to RQC targeting. We focused our analysis on two poorly understood 80S ribosome-binding proteins previously implicated in the response to failed translation, Asc1 and Hel2, as well as a new component of the pathway, Slh1, that we identified here. We found that premature arrest at ribosome stalling sequences still occurred robustly in the absence of Asc1, Hel2, and Slh1. However, these three factors were required for the RQC to modify the nascent chain. We propose that Asc1, Hel2, and Slh1 target arresting ribosomes and that this targeting event is a precondition for the RQC to engage the incomplete nascent chain and facilitate its degradation.

Stalled translation produces incomplete, ribosome-tethered polypeptides that the Ribosome-associated Quality Control (RQC) pathway targets for degradation via the E3 ubiquitin ligase Ltn1. During this process, the protein Rqc2 and the large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins can aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae . We found that CAT tails enhance Ltn1’s efficiency in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails to ubiquitylate those stalled polypeptides or becomes limiting, CAT tails act as degrons, marking proteins for proteasomal degradation off the ribosome. Thus, CAT tails functionalize the carboxy-termini of stalled polypeptides to drive their degradation on and off the ribosome.

Highlights d Longer forms of respiratory chain proteins accumulate under mitochondrial stress d Such proteins are formed by co-translational C-terminal extension (MISTERMINATE) d Such proteins can impair respiratory chain and also form cytosolic aggregates d MISTERMINATE links mitochondrial dysfunction with proteostasis failure in disease

Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.

Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE. translation, attenuated disease progression (Johnson et al., 2013). These studies strongly support an intimate connection between mitochondria and cytosolic translation, although the underlying mechanism is poorly defined.Here we provide a molecular link between mitochondrial function and cytosolic translation/proteostatsis. Mitochondrial damage induces translational stalling of mitochondrial outer membrane (MOM)-associated C-I30 mRNA by impairing the termination and ribosomerecycling factors eRF1 and ABCE1. Stalled ribosomes continue to add certain AAs to the Cterminus of C-I30, in a process analogous to CAT-tailing but with distinct features. C-I30 with C-terminal extension (CTE) inhibits oxidative phosphorylation (OxPhos) when assembled into C-I, and disrupts proteostasis when aggregating in the cytosol. Genetic manipulations of eRF1, ABCE1, or conserved CAT-tailing machinery prevent C-I30 CTE formation and rescue neuromuscular degeneration in PINK1 flies. PINK1/Parkin mechanistically regulates the RQC and CTE processes. These results identify C-I30 and possibly ATP5a as the first endogenous metazoan substrates of the CTE process, revealing intimate connections between mitochondrial health, co-translational QC, and proteostasis in neuromuscular tissues. RESULTS A Novel Form of C-I30 in PINK1 Neuromuscular TissuesHuman C-I is the largest enzyme of the respiratory chain, comprising 45 nuclear-or mitochondrial-encoded subunits, with C-I30 being part of the core assembly (Formosa et al., 2018). Mutations in C-I30 cause OxPhos and neuromuscular defects in Leigh syndrome. C-I30mRNA is translationally repressed in the cytosol before being recruited to MOM and reactivated by PINK1 and Parkin (Gehrke et al., 2015), two factors linked to familial PD.In muscle and brain tissues of PINK1 flies, we detected canonical C-I30...

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasomemediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity. OPEN ACCESS Citation: Sitron CS, Park JH, Giafaglione JM, Brandman O (2020) Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae. PLoS ONE 15(1): e0227841.

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks

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