# Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins

Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, Sae-Hun Park, Nikola Wagener, Anne Devin, Christof Osman, Walter Neupert, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, et al. Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins. Molecular Cell, 2025, 85 (21), pp.4082 - 4092.e7. 10.1016/j.molcel.2025.09.030. hal-05381437

HAL Id: hal-05381437

https://cnrs.hal.science/hal-05381437v1

Submitted on 25 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
**Molecular Cell**

# Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins

**Graphical abstract**

<figure><img src="image_3.png" /></figure>

**Authors**

Nils Bertram, Toshiaki Izawa,
Felix Thoma, ..., Christof Osman,
Walter Neupert, Dejana Mokranjac

**Correspondence**

mokranjac@bio.lmu.de

**In brief**

mitoRQC protects mitochondria against toxic CAT-tailed proteins. Using a genome-wide screen, Bertram et al. identify Pth2 as a peptidyl-tRNA hydrolase within mitoRQC. Pth2 modulates the import of CAT-tailed proteins and their access to the cytosolic QC network. Other hits from the screen show that, generally, delayed import protects mitochondria against toxic CAT-tailed proteins.

**Highlights**

* MitoRQC prevents the accumulation of toxic, mitochondria-localized CAT-tailed proteins

* A genome-wide screen identifies Pth2 as a peptidyl-tRNA hydrolase within mitoRQC

* Pth2 affects the import of CAT-tailed proteins and their access to the cytosolic QC network

* Delayed import, in general, protects mitochondria against toxic CAT-tailed proteins

<figure><img src="image_4.png" /></figure>
Short article

# Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins

Nils Bertram,<fnref n="1" /> Toshiaki Izawa,<fnref n="2" />,<fnref n="7" /> Felix Thoma,<fnref n="1" />,<fnref n="3" /> Serena Schwenkert,<fnref n="4" /> Stéphane Duvezin-Caubet,<fnref n="5" /> Sae-Hun Park,<fnref n="6" /> Nikola Wagener,<fnref n="2" /> Anne Devin,<fnref n="5" /> Christof Osman,<fnref n="1" /> Walter Neupert,<fnref n="2" /> and Dejana Mokranjac<fnref n="1" />,<fnref n="8" />,*

## SUMMARY

Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.

## INTRODUCTION

Defective proteins are constantly generated at a low rate during translation due to, e.g., mutations or damage to the mRNA or the ribosome. These aberrant nascent chains can aggregate, sequestering chaperones and inducing proteotoxic stress.<fnref n="1" />,<fnref n="2" /> To prevent protein toxicity at an early stage, the ribosome-associated protein quality control (RQC) system monitors and degrades translation products that emerge from stalled ribosomes.<fnref n="3" />⁻<fnref n="7" /> After dissociation of the stalled 80S ribosome,<fnref n="8" />⁻¹⁰ the 60S subunit, with associated peptidyl-tRNA, is recognized by Rqc2, which then recruits E3 ubiquitin ligase Ltn1.¹¹⁻¹⁴ Ltn1 ubiquitylates nascent chains at lysine residues present in close proximity to the ribosomal exit tunnel.¹²,¹³,¹⁵ Aided by Rqc1, together with Cdc48 and its cofactors, ubiquitylation leads to extraction of nascent chains and their subsequent degradation by the proteasome.¹¹,¹⁶,¹⁷ Rqc2 also has the ability to noncanonically elongate nascent chains by adding C-terminal alanine and threonine residues in a template- and 40S-independent manner.¹⁴,¹⁸⁻²¹ This modification, termed C-terminal alanyl and

threonyl (CAT) tailing, facilitates further ubiquitylation by Ltn1 by extruding additional lysine residues from the ribosomal exit tunnel.²² CAT tails were shown to dislodge nascent chains from the ribosome catalytic centre²³ and serve as degrons in different organisms²⁴⁻²⁷; however, they also render proteins aggregation prone, leading to formation of SDS-insoluble aggregates.²⁰,²⁸⁻²⁰

RQC acting on stalled ribosomes engaged in synthesis of nuclear-encoded mitochondrial proteins is complicated by the presence of N-terminal mitochondrial targeting sequences (MTSs).²⁹ The MTSs can already engage with mitochondrial protein translocases, the translocase of the outer mitochondrial membrane (TOM) complex in the outer and the translocase of the inner mitochondrial membrane 23 (TIM23) complex in the inner membrane, and initiate translocation before the nascent chain is released from the 60S subunit. This results in apposition of 60S with the TOM complex, reducing Ltn1's access to the nascent chains and hindering their ubiquitylation and subsequent extraction and degradation in the cytosol.²⁹ However, CAT tailing can still occur, eventually resulting in the import of

<figure><img src="image_5.png" /></figure>

<footnote n="1">¹LMU Munich, Faculty of Biology, Cell Biology, 82152 Martinsried, Germany</footnote>

<footnote n="2">²LMU Munich, Faculty of Medicine, BMC-Cell Biology, 82152 Martinsried, Germany</footnote>

<footnote n="3">³Graduate School Life Science Munich, 82152 Martinsried, Germany</footnote>

<footnote n="4">⁴LMU Munich, Faculty of Biology, MSBioLMU, 82152 Martinsried, Germany</footnote>

<footnote n="5">⁵Université Bordeaux, CNRS, IBGC, UMR 5095, 33000 Bordeaux, France</footnote>

<footnote n="6">⁶Max Planck Institute of Biochemistry, Department of Cellular Biochemistry, 82152 Martinsried, Germany</footnote>

<footnote n="7">⁷Present address: Graduate School of Life Science, University of Hyogo, Ako-gun, Hyogo 678-1297, Japan</footnote>

<footnote n="8">⁸Lead contact</footnote>

<footnote>*Correspondence: mokranjac@bio.lmu.de</footnote>

<footnote>https://doi.org/10.1016/j.molcel.2025.09.030</footnote>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 1.** Pth2 is a peptidyl-tRNA hydrolase involved in yeast mitoRQC</figcaption></figure>

(A) Schematic of the synthetic genetic array analysis.

(B) Ten-fold serial dilutions of the indicated cells were spotted on plates containing fermentable (YPD) or non-fermentable carbon sources (YPG) and incubated at indicated temperatures.

(legend continued on next page)
CAT-tailed proteins into mitochondria.²⁵,²⁹ Mitochondria were recently shown to be especially sensitive to CAT-tailed proteins, leading to a collapse of the mitochondrial proteostasis network, compromising mitochondrial function, and ultimately resulting in cell death.²⁹ Therefore, mitochondria rely on the protective function of the cytosolic endonuclease Vms1,³¹ which acts as a safeguard by halting CAT tailing, through both peptidyl-tRNA cleavage³²,³³ and displacement of Rqc2 from the 60S subunit,²⁹,³⁴ leading to the release of aberrant—but non-CAT-tailed—proteins into mitochondria. This RQC pathway acting on the mitochondrial surface is termed mitochondrial RQC (mitoRQC).²⁹

To identify additional proteins involved in mitoRQC, we conducted genome-wide screens in yeast. We identified peptidyl-tRNA hydrolase 2 (Pth2), a functionally poorly characterized peptidyl-tRNA hydrolase in the mitochondrial outer membrane,³⁵⁻³⁷ as a release factor of CAT-tailed proteins arrested in the TOM complex. Data presented here indicate that Pth2 does not influence CAT tailing of nascent chains but rather their translocation into mitochondria, providing more time for the cytosolic proteostasis network to deal with the mitoRQC substrates. Deletions of nonessential TOM and TIM23 components had similar effects, suggesting that delaying protein translocation is a general mechanism protecting mitochondria against toxic CAT-tailed proteins.

## RESULTS AND DISCUSSION

### Pth2 is involved in mitoRQC

To identify novel proteins involved in mitoRQC, we employed genome-wide screens in the yeast *Saccharomyces cerevisiae*, looking for components able to restore growth of Δ*vms1Δltn1* cells²⁹ on non-fermentable medium at 37°C. A high-copy suppressor screen did not identify any obvious novel candidates, though *Ltn1* and *Vms1* were both repeatedly identified (data not shown). In the second approach, we mated Δ*vms1Δltn1* cells with the yeast deletion library and screened for triple mutants that are viable under the screening conditions (Figure 1A). *Rqc2* was present among the identified candidates, confirming the validity of the approach (Table S1). One candidate, *Pth2*, drew our attention as it was previously shown to be an outer mitochondrial membrane protein interacting with the TOM complex,³⁷ and its human homolog, *PTRH2*, was implicated in a number of human disorders.³⁸ To validate the results obtained in the screen, we deleted *PTH2* in wild-type (WT), Δ*vms1*, Δ*ltn1*, and Δ*vms1Δltn1* strains and conducted comprehensive growth analyses on fermentable (YPD) and non-fermentable (YPG) media. As in the screen, additional deletion of *PTH2* restored growth of Δ*vms1Δltn1* cells on both media, though not to the same extent as deletion of *RQC2* did (Figure 1B). We observed no growth defects in any of the single-deletion strains

nor in the Δ*ltn1Δpth2* and Δ*vms1Δpth2* double mutants under tested conditions (Figures 1B, S1A, and S1B).

Mitochondria contain an additional *Pth*, *Pth1*,³⁶ whose mammalian homolog, *PTRH1*, was recently implicated in the RQC pathway in mammalian cells.³³ In addition, bacterial *Pth* was recently shown to release stalled nascent chains in a C-terminal-tail-dependent manner.²³ Interestingly, we did not identify *Pth1* in the screen. To exclude potential artefacts of high-throughput screening, we deleted *PTH1* in both WT and Δ*vms1Δltn1* cells. In contrast to *PTH2* deletion, deletion of *PTH1* did not improve growth of Δ*vms1Δltn1* cells but rather aggravated the already severe growth defect (Figures 1C and S1C). This observation can be explained by the different subcellular localizations of the two enzymes, supporting their involvement in different pathways. Whereas *Pth2* is integrated in the outer membrane and exposes its catalytic domain to the cytosol, *Pth1* is a soluble protein in the matrix (Figures 1D and 1E). Thus, *Pth2*, and not *Pth1*, is involved in mitoRQC in yeast. Whether *PTRH1* localizes to the mitochondrial surface, under normal or stress conditions, remains to be determined.

Based on the primary sequence conservation, *Pth2* was previously divided into N- and C-terminal regions³⁵,³⁹ (Figure S1D). Though the N-terminal region lacks conservation on the primary sequence, it contains a predicted transmembrane domain in all examined orthologs (Figures S1D and S1E). The C-terminal region is highly conserved among different eukaryotic species (Figure S1D). To investigate involvement of the two regions within mitoRQC, we generated different constructs (Figures 1F, 1G, and S1F). The WT *Pth2* construct, expressed under the control of the *PTH2* promoter and genomically integrated in Δ*vms1Δltn1Δpth2* cells, reconstituted the Δ*vms1Δltn1* phenotype (Figures 1F, 1G, S1G, S1I, and S1K), validating the approach taken. The substitution of the entire C-terminal region of *Pth2* with superfolded GFP (*Pth2N-GFP*) led to a strain that phenocopied Δ*vms1Δltn1Δpth2*, suggesting that this construct is a functionally inactive form of *Pth2* (Figures 1F, S1F, S1G, and S1I). In contrast, the replacement of the transmembrane domain of *Pth2* with that of the mitochondrial outer membrane protein *Tom70* (*TMTom70-Pth2*), to maintain the localization of the C-terminal region of the protein, replicated the effect of expressing WT *Pth2*, suggesting that the C-terminal region of *Pth2* plays an essential role in mitoRQC (Figures 1F, S1F, S1G, and S1I). The construct lacking the transmembrane domain (*Pth2ΔTM*), which was previously shown to reside in the cytosol,³⁷ showed an intermediate phenotype (Figures 1F, S1F, S1G, and S1I).

To assess whether *Pth2* catalytic activity is important within mitoRQC, we introduced a point mutation (*Pth2D174N*) in its catalytic site that was previously demonstrated to impair the *Pth* activity of *Pth2*.³⁹ Δ*vms1Δltn1Δpth2* cells expressing *Pth2D174N* grew better
<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_22.png" /></figure>

(legend on next page)
than Δ*vms1Δltn1* cells (Figures 1G, S1D, S1F, S1H, and S1K), strongly suggesting that the Pth activity of Pth2 is toxic in Δ*vms1Δltn1* cells. To investigate whether this function is exclusive to Pth2 or whether another Pth could substitute for it, we fused Pth1 to the N-terminal region of Pth2 to ensure the localization of Pth1 in the outer membrane (Figures 1G and S1F). Δ*vms1Δltn1Δpth2* cells expressing Pth2N-Pth1 grew even worse than Δ*vms1Δltn1* cells (Figures 1G and S1K). However, a point mutant that abrogates the catalytic activity of Pth1 (Pth2N-Pth1^H24N)^33 improved cell growth (Figures 1G, S1F, S1J, and S1K).

Taken together, these data indicate that Pth2 is involved in mitoRQC pathway as a peptidyl-tRNA hydrolase. The function of Pth2 can, however, be substituted by another peptidyl-tRNA hydrolase, upon proper localization. This finding is in agreement with a previously demonstrated remarkable functional conservation of peptidyl-tRNA hydrolases—both Pth1 and Pth2 can substitute for the essential enzyme in bacteria.^36,39

**Absence of Pth2 reduces aggregate formation but does not affect CAT tailing**

Toxicity associated with mitoRQC dysfunction in Δ*vms1Δltn1* cells is primarily attributed to CAT-tail-dependent aggregation of proteins within the mitochondrial matrix.^29 Indeed, expression of mitochondrially targeted GFP variants extended with 10 or 15 AT repeats, to mimic CAT tails without initiating an RQC response, led to the formation of protein aggregates and inhibited growth of WT cells, especially with the longer CAT tail (Figures S2A–S2D). To investigate whether the absence of Pth2 mitigates protein aggregation, we isolated detergent-insoluble protein aggregates from the cell lysates of WT, Δ*vms1Δltn1*, Δ*vms1Δltn1Δpth2*, Δ*vms1Δltn1Δrqc2*, and Δ*vms1Δltn1Δpth2* cells with reintroduced Pth2. Rieske Fe-S protein Rip1, which was previously used as a marker for aggregation of mitochondrial proteins,^29 was present in the soluble fraction and was almost completely processed to its mature form in WT cells but was exclusively found in the aggregate fraction and was processed only to its intermediate form in Δ*vms1Δltn1* cells (Figure 2A), as described previously.^29 Whereas additional deletion of *RQC2* in Δ*vms1Δltn1Δrqc2* cells fully restored both the solubility and processing of Rip1, in Δ*vms1Δltn1Δpth2* cells only part of Rip1 was fully processed and soluble (Figure 2A). A fraction of the intermediate form of Rip1 was, however, also present in the soluble form. The precursor form of Rip1 did not increase in Δ*vms1Δltn1Δpth2* cells. In Δ*vms1Δltn1Δpth2* cells with reintroduced Pth2, Rip1 behaved essentially the same as

in Δ*vms1Δltn1*. Ssq1, an Hsp70 chaperone in the mitochondrial matrix, behaved similarly to Rip1. In contrast, cytosolic protein Pgk1 was always present in the soluble fraction. In agreement with these findings, assembly of ATP synthase and activity of the respiratory chain, which are essentially absent in Δ*vms1Δltn1*, were partially restored in Δ*vms1Δltn1Δpth2* cells (Figures S2E–S2H). Thus, additional deletion of *PTH2* partially improved solubility of mitochondrial proteins and restored mitochondrial physiology of Δ*vms1Δltn1* cells, correlating well with the observed partial growth rescue.

To test whether Pth2 affects CAT tailing, we expressed a mitochondrially targeted version of GFP lacking the stop codon (non-stop mitochondrially targeted GFP, NS-mtGFP) from an inducible promoter (Figure 2B). Translation of mRNAs lacking the stop codon results in proteins with a C-terminal poly-lysine stretch due to a partial translation of polyA tails. These poly-lysine stretches stall the NS proteins in the ribosomal exit tunnel, making them substrates of RQC.^40 CAT tailing of this construct, visible as a smear running slower than the monomeric GFP.^16,28,29 was obvious in cell extracts of Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2*. Furthermore, high-molecular-weight, SDS-insoluble aggregates were detected in the same samples in the stacking gel (Figure 2B). Neither CAT tailing nor aggregation of GFP was detected in WT or Δ*vms1Δltn1Δrqc2* cells. Therefore, Pth2 does not appear to majorly influence CAT tailing.

To further investigate the protein aggregates present in Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2* cells, we used wide-field fluorescence microscopy. Twenty hours after the induction of NS-mtGFP expression, only a faint GFP signal, colocalizing with mitochondrial tubules, was detected in WT and Δ*vms1Δltn1Δrqc2* cells (Figures 2C and S2I). In contrast, Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2* cells clearly contained GFP aggregates, which appeared smaller and less intense in the latter (Figure 2C). Quantification of number, size, and intensity of the aggregates (Figures 2D–2F) confirmed our assessments. Filter trap assay corroborated the presence of smaller amounts of SDS-insoluble aggregates in Δ*vms1Δltn1Δpth2* than in Δ*vms1Δltn1* cells (Figures S2J and S2K). The membrane-potential-sensitive dye TMRM^41 effectively stained mitochondria in the majority of WT and Δ*vms1Δltn1Δrqc2* cells, but not in cells with large GFP aggregates, indicating a breakdown of membrane potential (not quantified), in agreement with the mitochondrial physiology measurements. To assess the kinetics of aggregate formation in Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2* cells, we analyzed cells before, and 6, 12, 18, and 24 h after, NS-mtGFP induction. Whereas cells

**Figure 2. Absence of Pth2 reduces aggregate formation but does not affect CAT tailing**

(A) Total cell extracts (T) of cells grown in YPGal medium at 37°C were prepared and either directly analyzed by SDS-PAGE and western blot or first fractionated by centrifugation into supernatant (S), containing soluble proteins, and pellet (P), containing aggregated proteins. T and S, 10% of P. p., precursor; i, intermediate; and m, mature forms of Rip1.

(B) Scheme of the NS-mtGFP construct used (upper panel). Total cell extracts were prepared 20 h after induction of NS-mtGFP expression at 30°C and analyzed by SDS-PAGE and western blot. Stacking gel contains aggregated NS-mtGFP. Cat represents CAT tailed and ** an unclarified form of NS-mtGFP.

(C) Fluorescence microscopy of NS-mtGFP-expressing cells. Yeast cells were grown as in (B). Mitochondria were stained with TMRM. The GFP signal was scaled equally, and the TMRM signal was auto-adjusted.

(D–F) Quantification of microscopy images of Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2* cells from (C). Statistical analysis was performed by comparing the means of three biological replicates using a two-sided t test. Results are represented as mean with 95% confidence interval.

(G) Time-course fluorescence microscopy of Δ*vms1Δltn1* and Δ*vms1Δltn1Δpth2* cells upon induction of NS-mtGFP expression. Channels were adjusted as in (C). (H and I) Quantification of microscopy images from (G), performed as in (D)–(F). See also Figure S2.
before induction had no visible GFP signal, aggregates were visible in both strains 6 h post induction. The aggregates were smaller and less intense in $\Delta vms1\Delta ltn1\Delta pth2$ than in $\Delta vms1\Delta ltn1$ cells (Figure 2G). Overall, $\Delta vms1\Delta ltn1$ cells showed a time-dependent decrease in number of aggregates per cell, with a simultaneous increase in their intensity (Figures 2H and 2I). The number of aggregates per cell consistently remained larger in $\Delta vms1\Delta ltn1\Delta pth2$ than in $\Delta vms1\Delta ltn1$ cells, and their intensity was comparably lower at all time points analyzed (Figures 2G–2I).

In summary, deletion of *PTH2* reduced aggregate formation in $\Delta vms1\Delta ltn1$ cells but did not majorly affect CAT tailing.

## Absence of Pth2 delays the import of CAT-tailed nascent chains

Building upon our finding that Pth2 functions as a Pth within mitoRQC, and on previous *in vitro* data indicating that Pth2 can release nascent chains from intrinsically destabilized 80S ribosomes<sup>42</sup> and PTRH2 from both 80S and 60S,<sup>33</sup> we analyzed whether NS-mtGFP-tRNA intermediates accumulate in the absence of Pth2 *in vivo*. In cell lysates prepared in the presence of an RNase inhibitor, we observed an additional GFP species migrating at approximately 50 kDa, which was weak in $\Delta vms1\Delta ltn1\Delta pth2$ cells and stronger in $\Delta vms1\Delta ltn1\Delta rqc2$ cells (Figure 3A). The same species was also present in $\Delta vms1\Delta pth2$ cells, but not in either of the single mutants (Figure S3A), suggesting partially overlapping functions of Vms1 and Pth2 and potentially explaining the poor growth of $\Delta vms1\Delta pth2$ cells upon overexpression of mitoRQC substrates (Figure S3B). Treatment with RNaseA completely removed this species, confirming its identity as peptidyl-tRNA.<sup>17</sup> Considering the difference in signal intensities of peptidyl-tRNA species in $\Delta vms1\Delta ltn1\Delta pth2$ and $\Delta vms1\Delta ltn1\Delta rqc2$ cells, it appears that, in the absence of both Vms1 and Pth2, another endonuclease and/or peptidyl-tRNA hydrolase can release nascent chains from the 60S subunit. However, neither Pth2 nor this additional enzyme seems to have access to non-CAT-tailed peptidyl-tRNAs, which accumulate in $\Delta vms1\Delta ltn1\Delta rqc2$ cells. Accumulation of peptidyl-tRNAs in $\Delta vms1\Delta ltn1\Delta pth2$ cells is likely underestimated here as it is unclear to what extent this species accumulates in the aggregate fraction. Strong accumulation of peptidyl-tRNAs observed in $\Delta vms1\Delta ltn1\Delta rqc2$ cells may explain why deletion of *RQC2* did not restore growth of $\Delta vms1\Delta ltn1$ cells upon induction of NS-mtGFP expression (Figure S3C)—CAT tailing may be crucial for ribosome recycling in the presence of overwhelming amounts of RQC substrates.

We next compared the interactomes of NS-mtGFP in $\Delta vms1\Delta ltn1\Delta pth2$ and $\Delta vms1\Delta ltn1$ cells. For this, we analyzed NS-mtGFP immunoprecipitates from the two strains by mass spectrometry (Figure 3B; Table S2). 60S ribosomal proteins predominantly clustered on the $\Delta vms1\Delta ltn1\Delta pth2$ side of the volcano plot, with 10%-30% enrichments indicating more stable interaction of NS-mtGFP with 60S in $\Delta vms1\Delta ltn1\Delta pth2$ cells. This finding is consistent with a reduced release of stalled nascent chains from the 60S subunit in the absence of Pth2. Moreover, the interactome of NS-mtGFP in $\Delta vms1\Delta ltn1\Delta pth2$ cells contained reduced levels of the majority of mitochondrial proteins—predominantly matrix proteins, including the chaperones Hsp60 and mtHsp70 (Figures 3B and 3C)—consistent

with their decreased aggregation in these cells (Figures 2A–2F). One notable exception was Tom40, which was enriched in the interactome in $\Delta vms1\Delta ltn1\Delta pth2$ cells (Figure 3B). Increased association of NS-mtGFP with Tom40 in $\Delta vms1\Delta ltn1\Delta pth2$ cells, compared with $\Delta vms1\Delta ltn1$ cells, was confirmed by western blot (Figure 3C). Among the proteins with the highest enrichment in the NS-mtGFP interactome in $\Delta vms1\Delta ltn1\Delta pth2$ cells was Sis1 (Figure 3B), the cytosolic chaperone previously shown to interact with cytosolic CAT-tailed proteins.<sup>18,28,43</sup> We also validated this result using western blot (Figure 3C). Although Sis1 aggregation occurred in both $\Delta vms1\Delta ltn1$ and $\Delta vms1\Delta ltn1\Delta pth2$ cells, aggregated Sis1 was specifically bound to NS-mtGFP only in $\Delta vms1\Delta ltn1\Delta pth2$ cells (Figure 3C). These data indicate that the stalled nascent chains dwell longer in the TOM complex and have increased access to the cytosolic proteostasis network in $\Delta vms1\Delta ltn1\Delta pth2$ cells compared with $\Delta vms1\Delta ltn1$ cells.

## Delayed translocation protects mitochondria against toxic CAT-tailed proteins

Our data suggest that the NS-mtGFP-tRNA intermediate bound to 60S is stabilized in the TOM pore in $\Delta vms1\Delta ltn1\Delta pth2$ cells, preventing its complete translocation. We therefore wondered whether delayed translocation of CAT-tailed proteins could be a general protective mechanism. To this end, we re-analyzed the list of candidates obtained in the screen, and, indeed, virtually all nonessential subunits of the TOM and TIM23 complexes were among the genes whose deletion restored the growth of $\Delta vms1\Delta ltn1$ cells (Table S1). We confirmed that additional deletions of *TOM6* from the TOM complex and *TIM21* from the TIM23 complex restored growth of $\Delta vms1\Delta ltn1$ cells essentially to WT levels on fermentable and non-fermentable media and at 30°C and 37°C (Figures 4A and 4B).

Based on the available data, we propose an updated model of mitoRQC (Figure 4C). Substrates of mitoRQC are faulty, nuclear-encoded mitochondrial proteins that are still bound to the 60S ribosomal subunit and accumulate in the TOM and TIM23 complexes on their way into mitochondria. In the absence of Vms1 and Ltn1, they are constantly CAT tailed by Rqc2. These CAT-tailed nascent chains are substrates of Pth2 in the outer membrane. Pth2, whose catalytic domain is connected to the outer membrane via a long and flexible linker (Figure S4), releases CAT-tailed nascent chains from the 60S subunit, allowing their import into mitochondria. However, once in the mitochondrial matrix, these CAT-tailed proteins aggregate, sequestering chaperones and resulting in mitochondrial toxicity. In the absence of Pth2, the release of CAT-tailed nascent chains is delayed and they remain longer in the TOM complex. Longer dwelling time in the TOM complex, likely accompanied by increased movement of the nascent chain within the translocation channel, provides more time and accessibility for the components of the cytosolic proteostasis network—including both the ubiquitin-proteasome system and chaperones such as Sis1—to act. The prolonged access of mitoRQC substrates to the cytosolic proteostasis network can also be achieved by lowering the activities of the TOM and TIM23 complexes. At the same time, delayed import of CAT-tailed proteins reduces their aggregation inside mitochondria, relieving the organellar proteostasis network and restoring mitochondrial function.
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 3. Absence of Pth2 delays import of CAT-tailed nascent chains**</figcaption></figure>

<figure><img src="image_25.png" /></figure>

(A) Analysis of tRNA-linked NS-mtGFP. Cells were grown as in Figure 2B and lysed in either the presence of RNaseA or the RNase inhibitor DEPC. Lysates were analyzed by neutral pH SDS-PAGE and western blot. Arrow represents tRNA-NS-mtGFP conjugate and ** an unclarified form of NS-mtGFP.

(B) Volcano plot of NS-mtGFP interactors in Δ*vms1Δltn1Δpth2* vs. Δ*vms1Δltn1* cells. Yeast cells were grown as in Figure 2B. Lysates were immunoprecipitated with anti-GFP nanobodies followed by on-bead digestion and label-free quantification by mass spectrometry. 60S subunits are shown in blue and mitochondrial proteins in magenta.

(C) Cell lysates as in (B) were immunoprecipitated using anti-GFP nanobodies. Total (10%), non-bound (10%), and bound (100%) fractions were analyzed by SDS-PAGE and western blot with indicated antibodies.
See also Figure S3 and Table S2.

## Limitations of the study

The physiological role of Pth2 and its substrate specificity, particularly in comparison to Vms1, remain to be further explored. The interplay of Pth2 with Ltn1 requires further investigation. Moreover, it remains unclear which components of the cytosolic proteostasis network are directly involved in the extraction of stalled nascent chains from the TOM complex in Δ*vms1Δltn1Δpth2* cells. Subcellular localization of protein aggre-

gates in Δ*vms1Δltn1Δpth2* cells needs to be analyzed by higher-resolution techniques.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the lead contact, Dejana Mokranjac (mokranjac@bio.lmu.de).
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 4.** Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins</figcaption></figure>

(A and B) Growth of indicated yeast strains was analyzed as in Figure 1B.

(C) Updated model of mitoRQC. See text for details.

See also Figure S4.

**Materials availability**

Plasmids and yeast strains generated in this study will be available from the lead contact upon request.

**Data and code availability**

* The raw data of mass spectrometry were deposited in the PRIDE repository and are publicly available as of the date of publication under the accession number PRIDE: PXD057575.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

**ACKNOWLEDGMENTS**

We thank current and past members of the Mokranjac lab and Mitoclub for their support and stimulating discussions, Johannes Buchner for providing the antibody against Sis1, Roland Beckmann for critical comments, and F. Ulrich Hartl for his continuous support. We also thank Jochen Rech for his help with the original yeast screen and the Robotics Core Facility at the Max Planck Institute (MPI) of Biochemistry for support. This research was generously supported by the Deutsche Forschungsgemeinschaft (projects NE101/28-1 to W. N. and MO1944/2-1 and MO1944/3-1 to D.M.). N.B. is the recipient of a doctoral fellowship awarded by the Walter and Monika Neupert Foundation. F.T. and C.O. were supported by a Human Frontier Science Program research grant (RGP021/2023) and a Deutsche Forschungsgemeinschaft research

grant (OS410/3-1). Collaboration between D.M. and S.D.-C. was supported by the LMU-Bordeaux Research Cooperation Program and Bay-France (FK-21-2023). S.D.-C. and A.D. are supported by CNRS. We acknowledge the contributions of Walter Neupert, who passed away during the course of this work.

**AUTHOR CONTRIBUTIONS**

Conceptualization, D.M., W.N., and N.B.; methodology, D.M., N.B., F.T., S.S., S.D.-C., S.-H.P., and A.D.; resources, D.M., W.N., C.O., S.-H.P., and A.D.; investigation, N.B., T.I., S.S., N.W., and S.D.-C.; formal analysis, N.B., T.I., F.T., S.S., and S.D.-C.; visualization, N.B., T.I., F.T., and S.D.-C.; supervision, D.M., W.N., S.-H.P., and C.O.; funding acquisition, D.M., W.N., C.O., S.D.-C., and A.D.; writing – original draft, D.M. and N.B. All authors discussed the results obtained and commented on the manuscript.

**DECLARATION OF INTERESTS**

The authors declare no competing interests.

**STAR★METHODS**

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
* Yeast strains and plasmids

## METHOD DETAILS

* Synthetic genetic array analysis

* Analysis of growth of yeast cells

* Submitochondrial localization

* Total cell extracts

* Protein aggregation assay

* Filter trap assay

* Blue Native-PAGE

* Oxygen consumption assay

* Determination of enzymatic activities

* Peptidyl-tRNA accumulation assay

* Immunoprecipitation of NS-mtGFP

* LC-MS/MS analysis

* Fluorescence microscopy

* Pth2 antibody generation

## QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.molcel.2025.09.030.

Received: November 20, 2024

Revised: May 16, 2025

Accepted: September 26, 2025

Published: October 20, 2025

## REFERENCES

1. Pilla, E., Schneider, K., and Bertolotti, A. (2017). Coping with Protein Quality Control Failure. Annu. Rev. Cell Dev. Biol. 33, 439–465. https://doi.org/10.1146/annurev-cellbio-111315-125334.

2. Hipp, M.S., Kasturi, P., and Hartl, F.U. (2019). The proteostasis network and its decline in ageing. Nat. Rev. Mol. Cell Biol. 20, 421–435. https://doi.org/10.1038/s41580-019-0101-y.

3. Filbeck, S., Cerullo, F., Pfeffer, S., and Joazeiro, C.A.P. (2022). Ribosome-associated quality-control mechanisms from bacteria to humans. Mol. Cell 82, 1451–1466. https://doi.org/10.1016/j.molcel.2022.03.038.

4. Inada, T., and Beckmann, R. (2024). Mechanisms of Translation-coupled Quality Control. J. Mol. Biol. 436, 168496. https://doi.org/10.1016/j.jmb.2024.168496.

5. Iyer, K.V., Müller, M., Tittel, L.S., and Winz, M.-L. (2023). Molecular Highway Patrol for Ribosome Collisions. Chembiochem 24, e202300264. https://doi.org/10.1002/cbic.202300264.

6. Joazeiro, C.A.P. (2019). Mechanisms and functions of ribosome-associated protein quality control. Nat. Rev. Mol. Cell Biol. 20, 368–383. https://doi.org/10.1038/s41580-019-0118-2.

7. Howard, C.J., and Frost, A. (2021). Ribosome-associated quality control and CAT tailing. Crit. Rev. Biochem. Mol. Biol. 56, 603–620. https://doi.org/10.1080/10409238.2021.1938507.

8. Pisareva, V.P., Skabkin, M.A., Hellen, C.U.T., Pestova, T.V., and Pisarev, A.V. (2011). Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 1804–1817. https://doi.org/10.1038/emboj.2011.93.

9. Shao, S., von der Malsburg, K., and Hegde, R.S. (2013). Listerin-Dependent Nascent Protein Ubiquitination Relies on Ribosome Subunit Dissociation. Mol. Cell 50, 637–648. https://doi.org/10.1016/j.molcel.2013.04.015.

10. Shoemaker, C.J., Eyler, D.E., and Green, R. (2010). Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science 330, 369–372. https://doi.org/10.1126/science.1192430.

11. Defenouillère, Q., Yao, Y., Mouaikel, J., Namane, A., Galopier, A., Decourty, L., Doyen, A., Malabat, C., Saveanu, C., Jacquier, A., et al. (2013). Cdc48-associated complex bound to 60S particles is required

for the clearance of aberrant translation products. Proc. Natl. Acad. Sci. USA 110, 5046–5051. https://doi.org/10.1073/pnas.1221724110.

12. Lyumkis, D., Oliveira dos Passos, D., Tahara, E.B., Webb, K., Bennett, E. J., Vinterbo, S., Potter, C.S., Carragher, B., and Joazeiro, C.A.P. (2014). Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex. Proc. Natl. Acad. Sci. USA 111, 15981–15986. https://doi.org/10.1073/pnas.1413882111.

13. Shao, S., Brown, A., Santhanam, B., and Hegde, R.S. (2015). Structure and assembly pathway of the ribosome quality control complex. Mol. Cell 57, 433–444. https://doi.org/10.1016/j.molcel.2014.12.015.

14. Shen, P.S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M.H., Cox, J., Cheng, Y., Lambowitz, A.M., Weissman, J.S., et al. (2015). Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75–78. https://doi.org/10.1126/science.1259724.

15. Bengtson, M.H., and Joazeiro, C.A.P. (2010). Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470–473. https://doi.org/10.1038/nature09371.

16. Brandman, O., Stewart-Ornstein, J., Wong, D., Larson, A., Williams, C.C., Li, G.-W., Zhou, S., King, D., Shen, P.S., Weibezahn, J., et al. (2012). A Ribosome-Bound Quality Control Complex Triggers Degradation of Nascent Peptides and Signals Translation Stress. Cell 151, 1042–1054. https://doi.org/10.1016/j.cell.2012.10.044.

17. Verma, R., Oania, R.S., Kolawa, N.J., and Deshaies, R.J. (2013). Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome. eLife 2, e00308. https://doi.org/10.7554/eLife.00308.

18. Defenouillère, Q., Zhang, E., Namane, A., Mouaikel, J., Jacquier, A., and Fromont-Racine, M. (2016). Rqc1 and Ltn1 Prevent C-terminal Alanine-Threonine Tail (CAT-tail)-induced Protein Aggregation by Efficient Recruitment of Cdc48 on Stalled 60S Subunits. J. Biol. Chem. 291, 12245–12253. https://doi.org/10.1074/jbc.M116.722264.

19. Tesina, P., Ebine, S., Buschauer, R., Thoms, M., Matsuo, Y., Inada, T., and Beckmann, R. (2023). Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control. Mol. Cell 83, 607–621. e4. https://doi.org/10.1016/j.molcel.2023.01.020.

20. Yonashiro, R., Tahara, E.B., Bengtson, M.H., Khokhrina, M., Lorenz, H., Chen, K.-C., Kigoshi-Tansho, Y., Savas, J.N., Yates, J.R., Kay, S.A., et al. (2016). The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation. eLife 5, e11794. https://doi.org/10.7554/eLife.11794.

21. Osuna, B.A., Howard, C.J., Kc, S., Frost, A., and Weinberg, D.E. (2017). In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing. eLife 6, e27949. https://doi.org/10.7554/eLife.27949.

22. Kostova, K.K., Hickey, K.L., Osuna, B.A., Hussmann, J.A., Frost, A., Weinberg, D.E., and Weissman, J.S. (2017). CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides. Science 357, 414–417. https://doi.org/10.1126/science.aam7787.

23. Svetlov, M.S., Dunand, C.F., Nakamoto, J.A., Atkinson, G.C., Safdari, H. A., Wilson, D.N., Vázquez-Laslop, N., and Mankin, A.S. (2024). Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control. Mol. Cell 84, 715–726. e5. https://doi.org/10.1016/j.molcel.2023.12.002.

24. Sitron, C.S., and Brandman, O. (2019). CAT tails drive degradation of stalled polypeptides on and off the ribosome. Nat. Struct. Mol. Biol. 26, 450–459. https://doi.org/10.1038/s41594-019-0230-1.

25. Lv, L., Mo, J., Qing, Y., Wang, S., Chen, L., Mei, A., Xu, R., Huang, H., Tan, J., Li, Y., et al. (2024). NEMF-mediated Listerin-independent mitochondrial translational surveillance by E3 ligase Pirh2 and mitochondrial protease ClpXP. Cell Rep. 43, 113860. https://doi.org/10.1016/j celrep.2024.113860.

26. Lytvynenko, I., Paternoga, H., Thrun, A., Balke, A., Müller, T.A., Chiang, C. H., Nagler, K., Tsaprailis, G., Anders, S., Bischofs, I., et al. (2019). Alanine
Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control. Cell 178, 76-90.e22. https://doi.org/10.1016/j.cell.2019.05.002.

27. Thrun, A., Garzia, A., Kigoshi-Tansho, Y., Patil, P.R., Umbaugh, C.S., Dallinger, T., Liu, J., Kreger, S., Patrizi, A., Cox, G.A., et al. (2021). Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing. Mol. Cell 81, 2112-2122.e7. https://doi.org/10.1016/j.molcel.2021.03.004.

28. Choe, Y.-J., Park, S.-H., Hassemer, T., Körner, R., Vincenz-Donnelly, L., Hayer-Hartl, M., and Hartl, F.U. (2016). Failure of RQC machinery causes protein aggregation and proteotoxic stress. Nature 531, 191-195. https://doi.org/10.1038/nature16973.

29. Izawa, T., Park, S.-H., Zhao, L., Hartl, F.U., and Neupert, W. (2017). Cytosolic Protein Vms1 Links Ribosome Quality Control to Mitochondrial and Cellular Homeostasis. Cell 177, 890-903.e18. https://doi.org/10.1016/j.cell.2017.10.002.

30. Wu, Z., Tantray, I., Lim, J., Chen, S., Li, Y., Davis, Z., Sitron, C., Dong, J., Gispert, S., Auburger, G., et al. (2019). MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure. Mol. Cell 75, 835-848.e8. https://doi.org/10.1016/j.molcel.2019.06.031.

31. Heo, J.-M., Livnat-Levanon, N., Taylor, E.B., Jones, K.T., Dephoure, N., Ring, J., Xie, J., Brodsky, J.L., Madeo, F., Gygi, S.P., et al. (2010). A stress-responsive system for mitochondrial protein degradation. Mol. Cell 40, 465-480. https://doi.org/10.1016/j.molcel.2010.10.021.

32. Verma, R., Reichermeier, K.M., Burroughs, A.M., Oania, R.S., Reitsma, J. M., Aravind, L., and Deshaies, R.J. (2018). Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes. Nature 557, 446-451. https://doi.org/10.1038/s41586-018-0022-5.

33. Kuroha, K., Zinoviev, A., Hellen, C.U.T., and Pestova, T.V. (2018). Release of Ubiquitinated and Non-ubiquitinated Nascent Chains from Stalled Mammalian Ribosomal Complexes by ANKZF1 and Pthr1. Mol. Cell 72, 286-302.e8. https://doi.org/10.1016/j.molcel.2018.08.022.

34. Su, T., Izawa, T., Thoms, M., Yamashita, Y., Cheng, J., Berninghausen, O., Hartl, F.U., Inada, T., Neupert, W., and Beckmann, R. (2019). Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis. Nature 570, 538-542. https://doi.org/10.1038/s41586-019-1307-z.

35. De Pereda, J.M., Waas, W.F., Jan, Y., Ruoslahti, E., Schimmel, P., and Pascual, J. (2004). Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity. J. Biol. Chem. 279, 8111-8115. https://doi.org/10.1074/jbc.M311449200.

36. Rosas-Sandoval, G., Ambrogelly, A., Rinehart, J., Wei, D., Cruz-Vera, L.R., Graham, D.E., Stetter, K.O., Guarneros, G., and Söll, D. (2002). Orthologs of a novel archaeal and of the bacterial peptidyl-tRNA hydrolase are nonessential in yeast. Proc. Natl. Acad. Sci. USA 99, 16707-16712. https://doi.org/10.1073/pnas.222659199.

37. Schulte, U., den Brave, F., Haupt, A., Gupta, A., Song, J., Müller, C.S., Engelke, J., Mishra, S., Mårtensson, C., Ellenrieder, L., et al. (2023). Mitochondrial complexome reveals quality-control pathways of protein import. Nature 614, 153-159. https://doi.org/10.1038/s41586-022-05641-w.

38. Sharkia, R., Jain, S., Mahajnah, M., Habib, C., Azem, A., Al-Shareef, W., and Zalan, A. (2023). PTRH2 Gene Variants: Recent Review of the Phenotypic Features and Their Bioinformatics Analysis. Genes 14, 1031. https://doi.org/10.3390/genes14051031.

39. Ishii, T., Funakoshi, M., and Kobayashi, H. (2006). Yeast Pth2 is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway. EMBO J. 25, 5492-5503. https://doi.org/10.1038/sj.emboj.7601418.

40. Joazeiro, C.A.P. (2017). Ribosomal Stalling During Translation: Providing Substrates for Ribosome-Associated Protein Quality Control. Annu. Rev. Cell Dev. Biol. 33, 343-368. https://doi.org/10.1146/annurev-cellbio-111315-125249.

41. Chazotte, B. (2011). Labeling Mitochondria with MitoTracker Dyes. Cold Spring Harb. Protoc. 2011, 990-992. https://doi.org/10.1101/pdb.prot5648.

42. Ito, Y., Yadani, Y., Niwa, T., Yamakawa, A., Machida, K., Imataka, H., and Taguchi, H. (2022). Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes. Nat. Commun. 13, 7451. https://doi.org/10.1038/s41467-022-35156-x.

43. Chang, W.D., Yoon, M.-J., Yeo, K.H., and Choe, Y.-J. (2024). Threonine-rich carboxyl-terminal extension drives aggregation of stalled polypeptides. Mol. Cell 84, 4334-4349.e7. https://doi.org/10.1016/j.molcel.2024.10.011.

44. Stan, T., Ahting, U., Dembowski, M., Künkele, K.P., Nussberger, S., Neupert, W., and Rapaport, D. (2000). Recognition of preproteins by the isolated TOM complex of mitochondria. EMBO J. 19, 4895-4902. https://doi.org/10.1093/emboj/19.18.4895.

45. Genge, M.G., Roy Chowdhury, S., Dohnálek, V., Yunoki, K., Hirashima, T., Endo, T., Doležal, P., and Mokranjac, D. (2023). Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes. Life Sci. Alliance 6, e202302122. https://doi.org/10.26508/lsa.202302122.

46. Banerjee, R., Gladkova, C., Mapa, K., Witte, G., and Mokranjac, D. (2015). Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein. eLife 4, e11897. https://doi.org/10.7554/eLife.11897.

47. Wagener, N., Ackermann, M., Funes, S., and Neupert, W. (2011). A pathway of protein translocation in mitochondria mediated by the AAA-ATPase Bcs1. Mol. Cell 44, 191-202. https://doi.org/10.1016/j.molcel.2011.07.036.

48. Jores, T., Lawatscheck, J., Beke, V., Franz-Wachtel, M., Yunoki, K., Fitzgerald, J.C., Macek, B., Endo, T., Kalbacher, H., Buchner, J., et al. (2018). Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins. J. Cell Biol. 217, 3091-3108. https://doi.org/10.1083/jcb.201712029.

49. Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., et al. (2004). A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947-962. https://doi.org/10.1002/yea.1142.

50. Houseley, J., and Tollervey, D. (2011). Repeat expansion in the budding yeast ribosomal DNA can occur independently of the canonical homologous recombination machinery. Nucleic Acids Res. 39, 8778-8791. https://doi.org/10.1093/nar/gkr589.

51. Longtine, M.S., McKenzie, A., Demarini, D.J., Shah, N.G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J.R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961. https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U.

52. Popov-Čeleketić, D., Waegemann, K., Mapa, K., Neupert, W., and Mokranjac, D. (2011). Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex. EMBO Rep. 12, 542-548. https://doi.org/10.1038/embor.2011.72.

53. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682. https://doi.org/10.1038/nmeth.2019.

54. Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367-1372. https://doi.org/10.1038/nbt.1511.

55. Tyanova, S., Temu, T., and Cox, J. (2016). The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301-2319. https://doi.org/10.1038/nprot.2016.136.
56. Hallgren, J., Tsirigos, K.D., Pedersen, M.D., Almagro Armenteros, J.J.A., Marcatili, P., Nielsen, H., Krogh, A., and Winther, O. (2022). DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. Preprint at bioRxiv. https://doi.org/10.1101/2022.04.08.487609.

57. Schrott, S., and Osman, C. (2023). Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae. Nucleic Acids Res. 51, 11813–11835. https://doi.org/10.1093/nar/gkad849.

58. Schreiner, B., Westerburg, H., Forné, I., Imhof, A., Neupert, W., and Mokranjac, D. (2012). Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria. Mol. Biol. Cell 23, 4335–4346. https://doi.org/10.1091/mbc.E12-05-0420.

59. Tong, A.H.Y., and Boone, C. (2007). 16 High-Throughput Strain Construction and Systematic Synthetic Lethal Screening in Saccharomyces cerevisiae. In Methods in Microbiology Yeast Gene Analysis, I. Stansfield and M.J. Stark, eds. (Academic Press), pp. 369–707. https://doi.org/10.1016/S0580-9517(06)36016-3.

60. Tong, A.H., Evangelista, M., Parsons, A.B., Xu, H., Bader, G.D., Pagé, N., Robinson, M., Raghibizadeh, S., Hogue, C.W., Bussey, H., et al. (2001). Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368. https://doi.org/10.1126/science.1065810.

61. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H., and Wanker, E. E. (1997). Huntingtin-Encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo. Cell 90, 549–558. https://doi.org/10.1016/S0092-8674(00)80514-0.

62. Bouchez, C.L., Hammad, N., Cuvellier, S., Ransac, S., Rigoulet, M., and Devin, A. (2020). The Warburg Effect in Yeast: Repression of Mitochondrial Metabolism Is Not a Prerequisite to Promote Cell Proliferation. Front. Oncol. 10, 1333. https://doi.org/10.3389/fonc.2020.01333.

63. Rappsilber, J., Ishihama, Y., and Mann, M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670. https://doi.org/10.1021/ac026117i.

64. Espinoza-Corral, R., Schwenkert, S., and Schneider, A. (2023). Characterization of the preferred cation cofactors of chloroplast protein kinases in Arabidopsis thaliana. FEBS Open Bio 13, 511–518. https://doi.org/10.1002/2211-5463.13563.

65. Cox, J., Hein, M.Y., Luber, C.A., Paron, I., Nagaraj, N., and Mann, M. (2014). Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526. https://doi.org/10.1074/mcp.M113.031591.

66. RStudio Team (2019). RStudio: Integrated Development for R, Version 1.2.5001 (RStudio, Inc.).

67. Bolte, S., and Cordelières, F.P. (2006). A guided tour into subcellular co-localization analysis in light microscopy. J. Microsc. 224, 213–232. https://doi.org/10.1111/j.1365-2818.2006.01706.x.

68. Lord, S.J., Velle, K.B., Mullins, R.D., and Fritz-Laylin, L.K. (2020). SuperPlots: Communicating reproducibility and variability in cell biology. J. Cell Biol. 219, e202001064. https://doi.org/10.1083/jcb.202001064.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Rabbit anti-Pth2</td><td>This study</td><td>Pth2, Rabbit 3, affinity purified</td></tr><tr><td>Mouse monoclonal anti-GFP</td><td>Roche</td><td>Cat. #11814460001; RRID:AB_390913</td></tr><tr><td>Rabbit anti-Tom70</td><td>Stan et al.<sup>44</sup></td><td>Rabbit 312</td></tr><tr><td>Rabbit anti-Tom40</td><td>Genge et al.<sup>45</sup></td><td>Rabbit 547, affinity purified</td></tr><tr><td>Rabbit anti-Tim23C-term</td><td>Neupert lab antibodies</td><td>Tim23C-pep, affinity purified</td></tr><tr><td>Rabbit anti-Tim44</td><td>Banjeree et al.<sup>46</sup></td><td>Rabbit 388, affinity purified</td></tr><tr><td>Rabbit anti-Rip1</td><td>Wagener et al.<sup>47</sup></td><td>Rip1C-pep, affinity purified</td></tr><tr><td>Rabbit anti-Ssq1</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>Mouse monoclonal anti-Pgk1</td><td>Life Technologies</td><td>Cat. #459250</td></tr><tr><td>Rabbit anti-F1β</td><td>Izawa et al.<sup>29</sup></td><td>Rabbit 421</td></tr><tr><td>Rabbit anti-Porin</td><td>Neupert lab antibodies</td><td>Rabbit 87118</td></tr><tr><td>Rabbit anti-Sis1</td><td>Jores et al.<sup>48</sup></td><td>N/A</td></tr><tr><td>Goat Anti-Mouse IgG (H+L)-HRP Conjugate</td><td>Bio-Rad</td><td>Cat. #1706516; RRID:AB_11125547</td></tr><tr><td>Goat Anti-Rabbit IgG (H+L)-HRP Conjugate</td><td>Bio-Rad</td><td>Cat. #1706515, RRID:AB_11125142</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>5-Fluoroorotic acid (5-FOA)</td><td>US Biological Life Sciences</td><td>Cat. #F5050</td></tr><tr><td>Bacto Peptone Gibco</td><td>Otto Nordwald</td><td>Cat. #211830</td></tr><tr><td>Bacto yeast Extract</td><td>Otto Nordwald</td><td>Cat. #212730</td></tr><tr><td>Yeast Nitrogen Base</td><td>Otto Nordwald</td><td>Cat. #291920</td></tr><tr><td>(S)-Lactic acid</td><td>MERCK</td><td>Cat. # 100366</td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Sigma-Aldrich</td><td>Cat. #T6399</td></tr><tr><td>Triton X-100</td><td>VWR</td><td>Cat. #M143</td></tr><tr><td>Digitonin</td><td>Calbiochem</td><td>Cat. # 300410</td></tr><tr><td>D(+)-Galactose</td><td>Sigma-aldrich</td><td>Cat. #48260</td></tr><tr><td>D(+)-Glucose</td><td>VWR</td><td>Cat. #24371.366</td></tr><tr><td>Glycerol</td><td>VWR</td><td>Cat. #24387.361</td></tr><tr><td>VeriFi® Polymerase</td><td>PCRBiosystems</td><td>Cat. #PB10.42-01</td></tr><tr><td>highQu ALLin™ Hifi DNA Polymerase</td><td>HighQu</td><td>Cat. # HLE0201</td></tr><tr><td>RibonucleaseA</td><td>ROTH</td><td>Cat. #9001-99-4</td></tr><tr><td>ChromoTek GFP-Trap® Magnetic Particles M-270</td><td>Proteintech</td><td>Cat. #AB_2827592</td></tr><tr><td>Sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)</td><td>ROTH</td><td>Cat. #P028-1</td></tr><tr><td>Glass beads 0.25–0.5 mm</td><td>ROTH</td><td>Cat. #A553.1</td></tr><tr><td>Urea</td><td>ROTH</td><td>Cat. #57-13-6</td></tr><tr><td>Phenylmethylsulfonylfluorid (PMSF)</td><td>Serva</td><td>Cat. #32395.04</td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>Cat. #43819</td></tr><tr><td>Dodecylsulfate-Na-salt (SDS)</td><td>Serva</td><td>Cat. #20765.03</td></tr><tr><td>Trizma® base (TRIS)</td><td>Sigma-Aldrich</td><td>Cat. #T1503-25G</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>Cat. #M3148</td></tr><tr><td>Complete protease inhibitor cocktail, EDTA-free</td><td>Roche</td><td>Cat. #05 056 489 001</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>Raw mass spectrometry data</td><td>This study</td><td>PRIDE: PXD057575</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Raw microscopy and picture data</td><td>This study</td><td>Mendeley Data: https://doi.org/10.17632/5vywz7sv39.1</td></tr></tbody></table>

Experimental models: Organisms/strains

<table><tr><td>BY4742</td><td>EUROSCARF</td><td>Y10000</td></tr><tr><td>BY4742 vms1Δ::kanMX4</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>BY4742 ltn1Δ::kanMX4</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>BY4742 pth2Δ::HIS3MX6</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 pth1Δ::natNT2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 pth2Δ::HIS3MX6 pth1Δ::natNT2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::kanMX4 pth2Δ::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 ltn1Δ::kanMX4 pth2Δ::HIS3MX6</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 rqc2Δ::LEU2</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth1Δ::natNT2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 tom6Δ::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 tim21Δ::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2N-GFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::TM<sub>Tom70</sub>-Pth2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2<sup>ΔTM</sup></td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2<sup>D174N</sup></td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2N-Pth1</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + LEU2::Pth2N-Pth1<sup>H24N</sup></td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::kanMX4 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 ltn1Δ::kanMX4 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 pth2Δ::HIS3MX6 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::kanMX4 pth2Δ::LEU2 + p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 ltn1Δ::kanMX4 pth2Δ::HIS3MX6 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 +p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 pth2Δ::LEU2 + p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 vms1Δ::HIS3MX6 ltn1Δ::kanMX4 rqc2Δ::LEU2 + p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr></table>

(Continued on next page)
**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>D273-10b</td><td>Neupert Lab strain collection</td><td>N/A</td></tr><tr><td>BY4742 Pth1-yEGFP</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 +pYes2</td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 +mtGFP<sub>AT10</sub></td><td>This study</td><td>N/A</td></tr><tr><td>BY4742 +mtGFP<sub>AT15</sub></td><td>This study</td><td>N/A</td></tr></tbody></table>

**Oligonucleotides**

<table><tr><td>Primers for cloning (see Table S3)</td><td>This study</td><td>N/A</td></tr></table>

**Recombinant DNA**

<table><tr><td>pSS036_Pth2_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_TM<sub>Tom70</sub>-Pth2_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_Pth2N-GFP_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_Pth2<sup>ΔTM</sup>_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_Pth2<sup>D174N</sup>_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_Pth2N-Pth1_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>pSS036_Pth2N-Pth1<sup>H24N</sup>_500bp</td><td>This study</td><td>N/A</td></tr><tr><td>p416_Gal1_NS-mtGFP</td><td>Izawa et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>p426_Gal1_NS-mtGFP</td><td>This study</td><td>N/A</td></tr><tr><td>pYM25</td><td>Janke et al.<sup>49</sup></td><td>P30237</td></tr><tr><td>pFA6a natNT2</td><td>Janke et al.<sup>49</sup></td><td>P30346</td></tr><tr><td>pFA6a LEU2</td><td>Houseley and Tollervey<sup>50</sup></td><td>#61923</td></tr><tr><td>pFA6a_His3MX6</td><td>Longtine et al.<sup>51</sup></td><td>#41596</td></tr><tr><td>pYes2</td><td>Popov-Celeketic et al.<sup>52</sup></td><td>Cat. #V82520</td></tr><tr><td>pYes2-mtGFP<sub>AT10</sub></td><td>This study</td><td>N/A</td></tr><tr><td>pYes2-mtGFP<sub>AT15</sub></td><td>This study</td><td>N/A</td></tr></table>

**Software and algorithms**

<table><tr><td>ImageJ 1.54p</td><td>Schindelin et al.<sup>53</sup></td><td>RRID:SCR_003070</td></tr><tr><td>R-studio</td><td>RStudio Team (2020)</td><td>N/A</td></tr><tr><td>MaxQuant 2.4.4.0</td><td>Cox and Mann<sup>54</sup></td><td>N/A</td></tr><tr><td>Perseus version 2.0.9.0</td><td>Tyanova et al.<sup>55</sup></td><td>N/A</td></tr><tr><td>Affinity Designer 1.10.6</td><td>https://affinity.serif.com/de/designer</td><td>N/A</td></tr><tr><td>SPECTROstar©Nano 5.70 R2</td><td>BMG Labtech</td><td>N/A</td></tr><tr><td>SPECTROstar©Nano MARS 4.01 R2</td><td>BMG Labtech</td><td>N/A</td></tr><tr><td>DeepTMHMM - 1.0</td><td>Hallgren et al.<sup>56</sup></td><td>RRID:SCR_025039</td></tr><tr><td>Jupyter Notebook 6.4.12</td><td>https://jupyter-notebook.readthedocs.io/en/6.4.12/</td><td>N/A</td></tr><tr><td>NIS-ELEMENTS AR 5.21.03</td><td>https://www.nisoftware.net/NikonSaleApplication/Default.aspx</td><td>N/A</td></tr><tr><td>Huygens essential 21.10.0p0 64b</td><td>https://svi.nl/Huygens-Essential</td><td>N/A</td></tr></table>

**Other**

<table><tr><td>NuPAGE™ 4-12% Bis-Tris-Gel</td><td>Thermo Fisher Scientific</td><td>Cat. #NP0322BOX</td></tr><tr><td>NativePAGE™ 3-12% Bis-Tris-Gel</td><td>Thermo Fisher Scientific</td><td>Cat. #BN1001BOX</td></tr><tr><td>Cellulose Acetate Membrane Circle (OE 66)</td><td>GE Healthcare</td><td>Cat. #10404131</td></tr></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### **Yeast strains and plasmids**

The wild-type *Saccharomyces cerevisiae* strain BY4742 (*Mat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0*) was used for all genetic manipulations. A comprehensive list of yeast strains and plasmids can be found in **STAR Methods key resource table** and of primers in **Table S3**.

Chromosomal tagging and deletions were made by homologous recombination of PCR products, as described previously.⁴⁹ The junctions were all confirmed by PCR and, if possible, also on protein level.
The various Pth2 constructs were genomically integrated into the *LEU* locus in a markerless manner, as described previously.⁵⁷ Briefly, DNA sequence encoding *PTH2*, together with ca. 500bp upstream and downstream of the ORF, was amplified from yeast genomic DNA and cloned into pSS036⁵⁷ via Gibson assembly, exchanging the DNA sequence encoding mitochondrially targeted mKate2 under the control of the *PGK1* promoter and the *ADH1* terminator with the *PTH2* sequence along with its promoter and 3'UTR. This plasmid served as a template for generation of all different Pth2 constructs using either Gibson assembly or mutagenesis PCRs. Before transformation into yeast, the plasmids were digested with NotI and transformants initially selected on selective glucose medium lacking uracil and subsequently on medium containing 5-FOA to remove the *URA* marker by recombination.⁵⁷

The construct p426Gal1-NS-mtGFP was created by digesting p416Gal1-NS-mtGFP²⁹ with Sacl and EcoRI and subcloning the fragment into Sacl/EcoRI digested pRS426.

pYES2-mtGFP<sub>AT10</sub> was generated by inserting the EcoRI/BamHI-digested GFP fragment from p416GAL1-mtGFP-(AT)<sub>10</sub>²⁹ into EcoRI/BamHI-digested pYES2, followed by addition of the matrix-targeting signal of yeast cytochrome *b*₂ (*b*₂(1-107)Δ19) as a HindIII/BamHI fragment cut out from pYes2-*b*₂(1-107)Δ19-DHFRmut.⁵⁸ pYES2-mtGFP<sub>AT15</sub> was subsequently created from pYES2-mtGFP<sub>AT10</sub> by mutagenesis PCR.

All plasmids were confirmed by Sanger sequencing.

Yeast cells were typically grown at 30°C in YP medium (10 g/L yeast extract, 20 g/L bactopeptone, pH 5.5) containing 2% of either glucose (YPD), galactose (YPGal), or glycerol (YPG), as a carbon source or in selective medium (1.7 g/L yeast nitrogen base, 5 g/L (NH₄)₂SO₄, 0.02 mg/mL adenine, 0.02 mg/mL histidine, 0.03 mg/mL leucine, 0.03 mg/mL lysine, 0.004 mg/mL tryptophan, pH 5.5) containing either 2% glucose (SD) or 2% lactate (SLac) as a carbon source.

Mitochondria were isolated from BY4742 Pth1-yEGFP and D273-10b cells grown in Lactate medium (3 g yeast extract, 1 g KH₂PO₄, 1 g NH₄Cl, 0.5 g CaCl₂x2H₂O, 0.5 g NaCl, 1.1 g MgSO₄x6H₂O, 0.3 ml 1% FeCl₃, 22 ml of 90 % lactic acid, dH₂O to 1 L, pH 5.5, supplemented with 0.1 % glucose) at 30°C, and BY4742, BY4742 Δ*vms1Δltn1*, BY4742 Δ*vms1Δltn1Δpth2* and BY4742 Δ*vms1Δltn1Δrqc2* grown in YPGal 37°C, as described in Genge et al. (2023).⁴⁵

## METHOD DETAILS

### Synthetic genetic array analysis

Synthetic genetic array analysis was performed essentially as described in Tong and Boone (2007).⁵⁹ Briefly, the MATa strain carrying query mutations (Δ*vms1::natNT2*, Δ*ltn1::hphNT1*) was constructed from the parental strain Y8205 (MATα Δ*can1*::*STE2pr-his5 Δlyp1*::*STE3pr-LEU2 ura3Δ0 leu2Δ0 his3Δ1 met15Δ0*)⁶⁰ and mated with the deletion mutant library (Thermo Fisher) using Biomek FXP (Beckman Coulter). After selection of diploid cells, sporulation was induced by transferring cells to sporulation medium containing 1% potassium acetate. The triple deletion mutants carrying Δ*vms1::natNT2*, Δ*ltn1::hphNT1* and XXXΔ::*kanMX4* were selected by transferring cells to the plates containing nourseothricin, hygromycin B and G418. The strains were then spotted onto the YPG plates, grown at 37°C for 3 days and their growth areas were measured. Each strain was generated in duplicates. Deletions with a z-score higher than 4.0 in both replicates are listed in Table S1.

### Analysis of growth of yeast cells

Growth of yeast cells was analyzed either by serial dilution spot assay on plates or in liquid media. For growth analysis on plates, yeast cells were grown in liquid YPD medium or in selective lactate medium containing 0.1% glucose ON at 30°C. ON cultures were diluted into fresh medium and grown until OD ca. 0.5. One OD of cells were then transferred into sterile tubes, cells were pelleted by centrifugation (16000 rcf, 2 min, RT) and resuspended in 1mL sterile dH₂O. Subsequently, four ten-fold serial dilutions were made in sterile dH₂O and 2 µl of cell suspensions were spotted on plates. The plates were incubated at indicated temperatures for 2 to 3 days.

Growth of yeast cells in liquid medium was analyzed in 96-well plates using the SPECTROstar Nano (BMG Labtech). To this end, ON cultures were diluted into fresh medium and grown till OD ca. 0.5. The cells were then collected, washed three times with sterile ddH₂O, resuspended in YPG medium to an OD₆₀₀ of 0.1 and aliquoted into wells of a 96-well plate. Growth at 30°C was monitored every 20 minutes for up to 72h under constant shaking at 800 rpm. All growth curves shown represent the mean of three biological replicates, each comprising technical triplicates. Calculations and visualizations were carried out using the SPECTROstar©Nano MARS 4.01 R2 software and Excel.

### Submitochondrial localization

Isolated mitochondria were resuspended in either SH buffer (0.6 M sorbitol, 20 mM HEPES/KOH, pH 7.4), to keep mitochondria intact, in 20 mM HEPES/KOH, pH 7.4, to burst the outer membrane and generate mitoplasts, or in 20 mM HEPES/KOH, pH 7.4 containing 0.25% Triton X-100, to solubilize mitochondrial membranes. Where indicated, proteinase K was added and the samples were incubated for 20min on ice. PMSF (1 mM) was then added to inhibit the protease and the samples were further incubated for 5 min on ice. Mitochondria and mitoplasts were reisolated by centrifugation (18000 rcf, 10 min, 4°C), resuspended in 2x Laemml buffer (120mM Tris/HCl, pH 6.8, 20% glycerol, 4% SDS, 0.02% bromphenol blue, 3% β-mercaptoethanol), incubated for 5 min at 95°C and then stored at -20°C until SDS-PAGE analysis. Triton X-100-solubilized sample was TCA-precipitated before resuspension in 2x Laemml buffer and analysis by SDS-PAGE and western blot.
To analyze membrane association of proteins by carbonate extraction, isolated mitochondria were incubated in 100 mM freshly prepared Na₂CO₃ for 30 min on a rotating platform at 4°C. One aliquot was taken as total and TCA-precipitated. The rest of the sample was centrifuged (30 min, 186000 rcf, 2°C) and thereby separated into the supernatant fraction, which contains the soluble and peripheral membrane proteins, and the pellet fraction which contains integral membrane proteins. The pellet fraction was directly resuspended in 2x Laemmli buffer whereas the supernatant was first TCA-precipitated. All samples were analyzed by SDS-PAGE and western blot.

**Total cell extracts**

Total cell extracts for analysis of CAT-tail proteins were prepared essentially as described in Izawa et al. (2017).²⁹ Briefly, expression of NS-mtGFP from the p426GAL1-NS-mtGFP plasmid was induced by addition of 0.5% galactose for 20h to logarithmically growing cells in selective lactate medium at 30°C. Cells (3 OD₆₀₀) were harvested by centrifugation (5000 rcf, 5 min, RT) and resuspended in 1 mL ice-cold H₂O before 150 µl of solution containing 2 M NaOH and 7 % β-mercaptoethanol were added. After 10 min incubation on ice, TCA was added to 12% final concentration and samples were incubated for an additional 30 min on ice. The precipitated proteins were isolated by centrifugation (17000 rcf, 10min, 2°C), washed with 1 mL ice-cold acetone and the final pellet was resuspended in 80 µl HU-buffer, incubated for 5 min at 95°C and analyzed by SDS-PAGE (1.5 OD₆₀₀ per lane) followed by western blot.

**Protein aggregation assay**

Endogenous protein aggregates were isolated essentially as previously described in Izawa et al. (2017).²⁹ Briefly, yeast cells were grown in YPGal medium ON at 37°C and diluted the next day into fresh medium. When the cultures reached OD ca. 0.5, 35 OD₆₀₀ of cells were collected by centrifugation (3220 rcf, 5min, RT), the pellets were resuspended in 500 µl IP buffer (50 mM Tris/HCl pH 7.4, 20 mM KCl, 10 mM MgCl₂, 1 mM PMSF, Protease inhibitor cocktail) and 100 µl glass beads were added. Cells were lysed by vortexing four times for 30s, with 30s pauses on ice in between, the samples were diluted with 500 µl IP buffer containing 1 % Triton X-100 and incubated for 10 min at 4°C with gentle rolling. After removal of the cell debris by centrifugation (840 rcf, 10min, 2°C), an aliquot corresponding to 5 OD₆₀₀ was taken as total (T) and the rest of the lysates were separated into soluble (S) and pellet (P) material by centrifugation (20000 rcf, 15min, 2°C). The pellets, containing aggregated proteins, were resolved in HU buffer (8 M urea, 5% SDS, 200 mM Tris/HCl pH 6.8, 1mM EDTA, 0.01% bromophenol blue, 2% β-mercaptoethanol), incubated for 5 min at 95°C and stored at -20°C until loading on SDS-PA gels. Proteins in T and S fractions were first TCA-precipitated (12% TCA), resolved in HU buffer, incubated for 5 min at 95°C and analyzed, together with the aggregate fraction, by SDS-PAGE followed by western blot. The material corresponding to 6 OD₆₀₀ was loaded per lane for P and 0.6 OD₆₀₀ per lane for T and S fractions.

**Filter trap assay**

Analysis of insoluble protein aggregates was performed as previously described (Izawa et al., 2017).²⁹ A total of 180 OD₆₀₀ of cells expressing NS-mtGFP for 20 hours were harvested and lysed in 1.2 mL of lysis buffer (25 mM Tris/HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 1 mM EDTA, 5% glycerol, 1x protease inhibitor tablet (Roche)) using a FastPrep-24 homogenizer equipped with a CoolPrep adaptor (MP Biomedicals). Lysates were clarified by centrifugation at 1,000 rcf for 10 minutes at 4 °C. Protein concentration in the lysates was determined using BCA assay (Bio-Rad) and specified amounts of proteins were either mixed 1:1 with 4% SDS, 100 mM DTT or with lysis buffer. SDS/DTT-treated samples were boiled at 95 °C for 3 minutes. All samples were then filtered through a 0.2 µm cellulose acetate membrane using a dot blot apparatus (Scherzinger et al., 1997).⁶¹ Membranes were immunodecorated with an anti-GFP antibody and visualized using HRP-labelled secondary antibodies and chemiluminescence. The signal intensities obtained with 200 µg SDS-treated samples were quantified using a standardized box and the “mean intensity” measurement functions in Fiji. Four biological replicates, each containing one replicate detected with the ImageQuant LAS 4000 mini (GE Healthcare) and one with Super RX X-ray film (Fujifilm), were analyzed. Mean values for each replicate were compared using a Student’s t-test function in Excel.

**Blue Native-PAGE**

Isolated mitochondria (5 mg/mL) were solubilized with 1.5 % digitonin in 50 mM NaCl, 50 mM imidazole, 1 mM EDTA, 1 mM PMSF, pH 7.0 on a rotating platform at 4°C for 10 min. Insoluble material was removed by centrifugation (150000 rcf, 15 min, 4°C), sample additive (ThermoFisher) and glycerol were added to the clarified extracts and samples were separated on 3-12% Native gels (ThermoFisher) according to the manufacturer's instructions. Gels were subsequently blotted on PVDF membranes and the membranes decorated with the indicated antibodies.

**Oxygen consumption assay**

Oxygen consumption was measured polarographically at 28°C using a Clark oxygen electrode (Chauvin-Arnoux) in a 1 mL thermostatically controlled chamber essentially as described before.⁶² Briefly, yeast cells were grown in YPGal medium at 37°C. Glycerol (10% (v/v)) was added to cultures in logarithmic growth phase, the cultures were frozen and kept at -80°C until use. Cells were thawed, washed, resuspended in fresh medium and transferred to the chamber (3-6 OD/mL). Respiratory rates were determined from the slope of O₂ concentration decrease over time. Measured activities were normalized per optical density unit at 600 nm and expressed in nano-atoms of Oxygen per minute and OD. Spontaneous respiratory rates were determined after addition of
100 mM ethanol. Uncoupled respiratory rates were measured after addition of 10 µM CCCP. Graphs show the means of four independent replicate measurements for the respective strains. Calculations and visualizations were performed in Excel.

## Determination of enzymatic activities

Cells, as detailed for oxygen consumption assay, were resuspended in 50 mM Tris/HCl pH 7.5, and broken by vigorous shaking (FastPrep MP Biomedicals, 4 m/s, 5 cycles 20s, 5min on ice in between) with an equal volume of glass beads. Unbroken cells and glass beads were removed by centrifugation. Protein concentration in cellular extracts was determined using the BCA (bicinchoninic acid) method (Pierce BCA Protein assay kit). Citrate synthase (EC2.3.3.1) activity was determined by monitoring the formation of coenzyme A, produced by citrate synthase from acetyl-CoA and oxaloacetate, by spectrophotometrically measuring the free sulfhydryl groups using Ellman's reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) over time at 412 nm, in a spectrophotometer (SAFAS, Monaco).⁶² Activity measurements were started by adding 10-100 µL of cellular extract to 50 mM Tris/HCl buffer pH 7.5 containing 0.05 % Triton-X100, 0.5 mM oxaloacetate, 0.1 mM acetyl-CoA, and 0.2 mM DTNB. When the signal increased linearly, the enzyme activity was calculated from the slope using an extinction coefficient of 13600 M⁻¹cm⁻¹ and expressed in nmoles of DTNB reduced per minute and mg protein. Cytochrome c oxidase activity (EC7.1.1.9; 5 mM potassium cyanide-sensitive) was determined by spectrophotometrically (550 nm) monitoring the rate of disappearance of reduced cytochrome c in the following buffer: 50 mM K-phosphate buffer, pH 7.5, 25 µM reduced cytochrome c.⁶² The assay was started by adding 10-40 µL of cellular extract. The enzyme activity was determined using an extinction coefficient of 18500 M⁻¹cm⁻¹ and expressed in nmoles of cytochrome c per minute and mg protein. Results shown are means of multiple measurements (5 to 12) from two to four separate samples. Calculations and visualizations were performed in Excel.

## Peptidyl-tRNA accumulation assay

To assess the presence of peptidyl-tRNAs in cells, the protocol described in Verma et al. (2013)¹⁷ was modified. Briefly, yeast cells (5 OD₆₀₀) were washed three times with 1 mL ice cold buffer containing 50 mM Tris/HCl, pH7.5, 10 mM NaN₃, 10 mM EDTA, 10 mM EGTA, 1x protease inhibitor tablet (Roche), 10 mM NEM, 50 mM NaF, 60 mM β-glycerophosphate, 10 mM sodium pyrophosphate, and split into two 200 µl aliquots. The cells were reisolated and 50 µl glass beads were added together with 40 µl SDS buffer (120 mM MOPS/NaOH, pH 6.8, 20 % glycerol, 4 % SDS, 0.02 % bromphenol blue) containing either 1 % DEPC and 2 % β-mercaptoethanol or 200 ng/µl RibonucleaseA. Cells were incubated at 65°C for 5 min and vigorously vortexed for 1 min. β-mercaptoethanol was added to 2% to RNaseA-treated samples. All samples were incubated for 3 min at 95°C, centrifuged for 1 min at 16000 rcf and directly loaded on 4-12 % NuPAGE gels (Thermo fisher). Gels were run in MOPS buffer (Thermo fisher) according to manufacturer's instructions and subsequently blotted on nitrocellulose membranes.

## Immunoprecipitation of NS-mtGFP

Cells (180 OD₆₀₀), 20h after expression of NS-mtGFP was induced, were collected by centrifugation (3220 rcf, 5min, RT) and resuspended in 600 µl lysis buffer (25 mM Tris/HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 1 mM EDTA, 5% glycerol, 1x protease inhibitor tablet (Roche)). Glass beads (50 µl) were added and the cells were vortexed four times for 30s with 30s pauses on ice. The samples were diluted with 600 µl lysis buffer containing 1% Triton X-100 and incubated for 10 min on a rolling platform at 4°C. After a clarifying spin (1000 rcf, 10 min, 4°C), 1 mL of lysate was added to 10 µl GFP-Trap® Magnetic Particles M-270 (Chromotek/Proteintech) and incubated for 2h at 4°C on a rotating platform. During this time, the remaining lysate was diluted with ice cold water to 1 mL and the proteins therein TCA-precipitated (12% TCA). GFP-Trap beads were isolated using a magnetic rack, the non-bound fraction was removed and the beads were subsequently washed three times for 10 min with 500 µl lysis buffer containing 0.05% Triton X-100. The specifically bound proteins were either directly eluted with HU buffer and incubation for 5 min at 95°C for analysis by SDS-PAGE and western blot or were directly used for on bead digest and mass spectrometry.

For mass spectrometry, three biological replicates of each strain were analyzed. The beads were washed twice with 50 mM Tris/HCl, pH 7.5, and subsequently with 50 mM Tris/HCl, pH 7.5 containing 2 M urea. For the first trypsin digest, the beads were resuspended in 160 µl of 50 mM Tris/HCl, pH 7.5, 1 M urea, 1 mM Dithiothreitol (DTT), and 0.8 µg of trypsin (Pierce, Thermo Scientific) were added and incubated for 3 h at 25°C with continuous shaking. Post-incubation, the supernatant was saved and the beads were washed twice with 120 µl 50 mM Tris/HCl, pH 7.5 containing 1 M urea. The supernatants were combined with the trypsin digest. To the pooled sample, DTT was added to a final concentration of 4 mM, and the sample was incubated at 25°C for 30 min with shaking. Iodoacetamide was added to a final concentration of 10 mM and the sample was incubated at 25°C for 45 min with shaking in the dark. Subsequently, an additional 1 µg of trypsin was added, and the second digestion was allowed to proceed overnight at 25°C with continuous shaking. The combined digested peptides were purified using home-made C18 stage tips.⁶³

## LC-MS/MS analysis

Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on a nano-LC system (Ultimate 3000) coupled to an Impact II Q-TOF (Bruker Daltonics, Bremen, Germany) using a CaptiveSpray nano electrospray ionization (ESI) source as described previously.⁶⁴ Peptides (1 µg) were separated over a 30 min linear gradient of 5–45% (v/v) acetonitrile on a Acclaim Pepmap RSLC analytical column (C18, 100 Å, 75 µm x 50 cm) (Thermo Scientific). MS1 spectra with a mass range of m/z 200–2000 were acquired
at 3 Hz and the 18 most intense peaks were selected for MS/MS analysis with an intensity-dependent spectrum acquisition rate be-
tween 4 and 16 Hz. Dynamic exclusion duration was set to 0.5 minutes.

Raw data files were analyzed using MaxQuant software version 2.4.4.0,⁵⁴ with peak lists compared against the *Saccharomyces cerevisiae* reference proteome from UniProt (www.uniprot.org). All settings were kept at default values and protein quantification was performed using the label-free quantification (LFQ) algorithm.⁶⁵ Further analysis was performed using Perseus version 2.0.9.0.⁵⁵ Potential contaminants, proteins identified only through site modification, and reverse hits were removed. Only proteins quantifiable by the LFQ algorithm in at least two out of three replicates were kept. LFQ intensities were transformed to log₂ values and missing data were imputed from a normal distribution within Perseus using standard parameters. Samples were statistically compared using a t-test and data was visualized using a custom RStudio⁶⁶ script.

Fluorescence microscopy

Yeast cells were grown in selective lactate medium containing 0.1% glucose ON at 30°C. On the next day, the cells were diluted into fresh selective lactate medium without any added sugar and grown till OD ca. 0.5. One sample per culture was directly imaged (0h) and the rest of the cultures were diluted to OD600 of 0.3 and expression of NS-mtGFP was induced by addition of galactose to 0.5%. If needed, the cultures were diluted in selective lactate medium containing 0.5% galactose so that OD600 remained between 0.3 and 0.8. After indicated time periods, 0.15 OD600 were isolated by centrifugation (3000 rcf, 3 min, RT), resuspended in 1 mL staining buffer (10 mM Hepes, pH 7.6, 5% glucose, sterile filtered) containing 50 nM Image-iT TMRM-Reagent (Thermo fisher) and rotated for 15 min at 30°C in the dark. Cells were re-isolated by centrifugation and washed two times with 1 mL staining buffer and once with 1 mL selective lactate medium containing 0.5% galactose. Cells were finally resuspended in 500 µl medium and 300 µl of the cell suspension were transferred into Concanavalin A coated 8-well chambered µ-slides with glass bottom (Ibidi). The cells were attached to Con A by a 2 min centrifugation of the slides at 800 rcf. Non-bound cells were subsequently washed three times with 400 µl medium. Images were captured in Z-stacks (41 slices, 200 nm) with a Nikon ECLIPSE Ti2-E microscope (Nikon MEA54000), with a CFI Apochromat TIRF 100XC oil immersion objective (Nikon MRD01991) and a Photometrics Prime 95B 25-mm camera (PH-P95B-25) at 30°C. The ND2 files of the NIS-ELEMENTS AR software (Nikon MQS31000) were converted to OME-TIFF using a custom Python 3 macro and deconvolved using Huygens Essential v 18.10 (Scientific Volume Imaging). Pictures in figures are presented as maximum intensity Z-projections created with Fiji/ImageJ. To allow for better visualization in case of the GFP channel pictures were equally and in case of the TMRM signal individually adjusted by setting the upper and lower borders of depicted pixel intensities. Single cells expressing GFP were manually cropped out using the Fiji ROI function and quantified with a custom Fiji/ImageJ macro. Briefly, the GFP channel of each cropped image was automatically analyzed for particles with the Fiji plugin "3D Objects Counter" ("slice=20 min.=7 max.=2562500 exclude_objects_on_edges objects surfaces statistics summary").⁶⁷ Initially, images were manually analyzed and evaluated to identify errors in the macro's performance. Subsequently, these erroneous analyses were automatically excluded by the macro using a predefined threshold. The data were then analyzed and plotted using custom Python scripts in Jupyter Notebook. Average values of three independent experiments with at least 150 cells per replicate were statistically compared using Student's t-test, as recommended by Lord et al. (2020)⁶⁸

Pth2 antibody generation

Antibodies against Pth2 was generated in rabbits by injection of recombinantly expressed and purified His~6~-Pth2(35-208). To validate the specificity of the obtained sera, total cell extracts from BY4742 and BY4742 *4pth2* yeast cells were separated by SDS-PAGE, transferred to nitrocellulose membranes, and the obtained stripes used for western blot using preimmune and the sera obtained after immunization of the rabbits. Pth2-specific antibodies were affinity-purified from the sera using an affinity column made by coupling the same His~6~-Pth2(35-208) fragment to CNBr-activated Sepharose 4B beads (Cytiva).

QUANTIFICATION AND STATISTICAL ANALYSIS

Details of the quantification and statistical analyses are provided in the respective STAR Methods sections, Figures and Fig-
ure legends. p-values are stated in the respective Figures. The antibody decorations as well as the serial dilution spot assays shown
are representative results of at least three biological replicates.