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Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles [1]

['Elena Bittner', 'Department Of Biology', 'Philipps-University Marburg', 'Marburg', 'Thorsten Stehlik', 'Jason Lam', 'Department Of Molecular', 'Cell Biology', 'Howard Hughes Medical Institute', 'University Of California']

Date: 2024-02

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER–mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Funding: TS received funding from a fellowship from DAAD ( https://www.daad.de/de/ ). GB thanks the European Research Council (ERC) for support through the project “KIWIsome” (Grant agreement number: 101019765). The project in the Schuldiner laboratory was supported by funding from ERC under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 864068). The robotic system of the Schuldiner laboratory was purchased through the support of the Blythe Brenden-Mann Foundation. MS is an Incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics. RS is an investigator of the Howard Hughes Medical Institute. JF was supported by a fellowship from Leopoldina and by the German research foundation (FR 3586/2-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Bittner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Organelle tethering has been a subject of intense research in recent years as it has become clear that organelles do not work in isolation but rather form areas of close apposition, contact sites, that enable the transfer of molecules and ions [ 14 ]. Tethering molecules that sustain such contacts have been discovered and, until now, have been shown to rely on components that either span the membrane or tightly bind to it [ 15 ]. Such a contact site is formed between peroxisomes and mitochondria and has been dubbed the PerMit [ 16 , 17 ]. Genetic data and protein interaction experiments reveal that the membrane proteins Pex11, Pex34, Fzo1, and the ERMES complex are important for peroxisome–mitochondria tethering [ 17 , 18 ]. Other tethers for peroxisomes to different organelles have been identified [ 19 – 25 ]. In mammalian cells, proximity of peroxisomes and the ER is achieved through interaction of membrane-associated proteins called VAMP-associated proteins A and B with a peroxisomal protein termed acyl-CoA binding domain-containing 5 [ 26 , 27 ]. Deletion of a single tether does not result in the loss of contact sites, suggesting that multiple tethering mechanisms are involved in the formation of each contact.

For many years, proteins were studied that either have a C-terminal PTS1 or an N-terminal mitochondrial targeting signal (MTS). Recently, it was shown that the protein phosphatase Ptc5 from the yeast Saccharomyces cerevisiae contains both targeting signals and exhibits a dual localization to peroxisomes and mitochondria [ 11 ]. Ptc5 is processed by the inner membrane peptidase (IMP) complex inside mitochondria and reaches the peroxisomal matrix via mitochondrial transit [ 11 , 12 ]. Additional proteins containing competing mitochondrial and peroxisomal targeting signals have been identified. Overexpression of several enhanced the number of peroxisomes proximal to mitochondria [ 11 ]. Data from mammals point to a similar phenomenon [ 13 ].

Translocation of peroxisomal matrix proteins requires distinct targeting signals termed peroxisome targeting signal type 1 and 2 (PTS1 and PTS2, respectively). PTS1 motifs are recognized and bound by the cytosolic targeting factor Pex5 and translocated via interaction of Pex5 with Pex13/14 on the peroxisomal membrane allowing the translocation of folded and even oligomerized protein complexes through Pex13, which acts as a pore resembling the nuclear import system [ 4 – 8 ]. Peroxisomes can multiply by growth and division, but unlike mitochondria can also form de novo in the absence of mature peroxisomes from the endoplasmic reticulum (ER) in a process which possibly involves mitochondria, at least in mammalian cells [ 4 , 9 , 10 ].

Metabolic pathways in eukaryotic cells are often compartmentalized inside membrane-enclosed organelles such as mitochondria and peroxisomes [ 1 ]. Proteins synthesized in the cytosol must target and translocate into their organelles of residence correctly, efficiently, and in a regulated manner [ 2 – 4 ]. Most mitochondrial proteins are imported from the cytosol in a conformationally flexible state via evolutionary conserved protein complexes called translocase of the outer membrane (TOM) and translocase of the inner mitochondrial membrane (TIM). Many proteins destined for the mitochondrial matrix or inner membrane contain N-terminal targeting signals that are cleaved by proteases upon import [ 3 ].

Results

Loss of the matrix protein targeting machinery reduces the number of contacts between peroxisomes and mitochondria If tethering is affected by dual targeting of MTS-PTS proteins, it should be reduced by eliminating the targeting factor Pex5. To test this, we quantified the number of peroxisomes associated with mitochondria in control and Δpex5 cells. A significant reduction of peroxisomes close to mitochondria was observed in the absence of Pex5 (Figs 3A, 3B, and S3) indicating a role of the peroxisomal matrix targeting machinery for the generation of contacts—something unexpected if only membrane proteins were required for tethering as is usually assumed. To further confirm this concept, we deleted PEX5 in a strain overexpressing Pxp2-RFP-PTS. Enhanced tethering triggered by Pxp2-RFP-PTS was blocked by deleting PEX5 (Fig 3C). In addition, we quantified the peroxisome number and detected an increase of smaller appearing peroxisomes upon deletion of PEX5 indicating that the reduced colocalization is not a result of a reduction in peroxisome number (Figs 3D and S3C). Another phenotype previously described for mutants with reduced peroxisome tethering is increased movement of the organelles [19,26]. Peroxisome movement was found to be enhanced in Δpex5 cells (S1 Movie). These results indicate that overexpression of dual affinity MTS-PTS proteins and depletion of Pex5 have a reciprocal effect on peroxisome number, morphology, and proximity to mitochondria. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Depletion of components of the peroxisomal import machinery reduces PerMit contacts. (A) Fluorescence microscopic picture of control and Δpex5 cells expressing endogenously tagged Pex3-GFP (green) and Tim50-RFP (magenta). White arrows denote peroxisomal signal overlapping with mitochondrial signal. (B) Quantification of the fraction of peroxisomes contacting mitochondria (Px M ) in relation to the total peroxisome count (Px T ) of control cells and Δpex5 cells. (C) Quantification of the fraction of peroxisomes contacting mitochondria (Px M ) in relation to the total peroxisome count (Px T ) of control cells and Δpex5 cells expressing Pxp2-RFP-PTS, Ant1-YFP, and Tim50-CFP. (D) The number of peroxisomes per cell was quantified in the indicated strains expressing Pex3-GFP. (E) Scheme of the genetic modifications used for auxin-dependent depletion of Pex13 in (F)–(I). The endogenous PEX13 locus was genetically engineered to encode a translational fusion of Pex13 with a C-terminal AID and 6 hemagglutinin (HA) tags. Pex13 degradation is mediated by the F-box protein AFB2 from Arabidopsis thaliana, which was expressed from the ADH1 promotor. (F) Auxin-dependent depletion of Pex13-AID-HA at indicated time points was analyzed by SDS-PAGE and immunoblot. Por1 served as a loading control. (G) Fluorescence microscopic images of indicated strains expressing the peroxisomal membrane protein Ant1-YFP (green) and RFP-PTS (magenta) in the absence (-Auxin) or presence (+Auxin; 4 h) of 2 mM indole-3-acetic acid. (H) Subcellular localization of Ant1-YFP (magenta) and Tim50-CFP (green) of indicated strains was analyzed in the presence of 2 mM indole-3-acetic acid at indicated time points (left). White arrows indicate peroxisomes in proximity to mitochondria. The fraction of peroxisomes in contact with mitochondria (Px M ) relative to the total peroxisome count (Px T ) of the indicated strain was quantified (right). (I) Identical to (H), except that the cells also expressed Cat2-RFP-HA-PTS (red) to increase PerMit contacts. Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. A one-way ANOVA combined with a Tukey test was performed to assess statistical significance for multiple comparisons. Otherwise, a two-sided unpaired Student’s t test was performed. Underlying data for quantifications can be found in S1 Data. AID, auxin-inducible degron; PTS, peroxisome targeting signal; RFP, red fluorescent protein. https://doi.org/10.1371/journal.pbio.3002508.g003 To confirm our findings and reduce the possibility that the observed phenotype only resulted from secondary, downstream effects, we took an approach for rapid depletion of the Pex5 receptor, Pex13 [6], using an auxin-inducible degron (AID) tag [35,36] (Fig 3E). Peroxisomal import of RFP-PTS was still visible upon AID-HA tagging of Pex13 demonstrating that the tagging itself does not abolish import. Addition of auxin lead to cytosolic accumulation of RFP-PTS (Figs 3G and S4A) and reduction of Pex13-AID-HA levels (Fig 3F). Association of peroxisomes and mitochondria was quantified in strains with or without Cat2-RFP-HA-PTS as an additional tether relative to control strains (Figs 3H, 3I, S4B–S4D, and S5). Both strains started with a different degree of organelle association but depletion of Pex13-AID-HA by auxin addition reduced the number of peroxisomes proximal to mitochondria to a similar basal level. These differences in tethering support a role of dual protein targeting in driving organelle association. To corroborate these data, we created a strain with conditional PEX5 expression by putting it under control of the GAL promoter (S6 Fig). Immunoblot and fluorescence microscopy of RFP-PTS expressing cells confirmed that Pex5 levels correlated with PTS import (S6A and S6B Fig). We then overexpressed Ptc5ΔTMD-RFP-PTS, a dually targeted derivative of Ptc5 lacking the transmembrane domain (TMD) that acts as a strong tether [11]. Automated time lapse imaging revealed that induction of Pex5 elevated the number of peroxisomes close to mitochondria, whereas its reduction had the opposite effect (S6C Fig). Since single planes were imaged, lower basal overlap of mitochondria and peroxisomes was observed in the absence of Pex5 (compare to Fig 3B). Together, these data suggest a function of peroxisomal matrix protein import for formation of PerMit contacts.

Screening for factors that regulate dual targeting of Ptc5 To identify additional factors that regulate dual targeting and therefore potentially also tethering, we conducted a high-content genetic screen focused on Ptc5 sorting from mitochondria to peroxisomes (Fig 5A). Peroxisomal import of Ptc5-RFP-PTS depends on mitochondrial processing and subsequent Pex5 dependent targeting [11]. This transit mechanism may require direct contact between the organelles. PPT PowerPoint slide

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TIFF original image Download: Fig 5. A high-content genetic screen to identify factors involved in targeting of Ptc5 to peroxisomes. (A) Schematic illustration of a high-content microscopic screen to uncover factors involved in sorting of the protein phosphatase Ptc5. A query strain co-expressing Ptc5-RFP-PTS and Pex3-GFP was crossed into indicated yeast libraries using synthetic genetic array technology. Haploid progeny expressing both fusion proteins in each mutant background were analyzed with automated fluorescence microscopy. MTS, mitochondrial targeting signal; TMD, transmembrane domain; PP2C, protein phosphatase type 2C; PTS1, peroxisomal targeting signal type 1. (B) Table showing a subset of mutants affecting Ptc5 localization. The entire list of mutations is depicted in S2 Table. (C) Subcellular localization of Ptc5-RFP-PTS (magenta) and Pex3-GFP (green) was analyzed in indicated strains using automated fluorescence microscopy. Shown are pictures from experiments performed on putative hits. Δcoq9 cells were used as a control as these show Ptc5-RFP-PTS localization in peroxisomes but are affected in mitochondrial metabolism [11]. Δpex5 mutants show mitochondrial but no peroxisomal signal of the reporter Ptc5-RFP-PTS. Scale bars represent 5 μm. (D) Correlation between Ptc5-RFP-PTS signal and Pex3-GFP signal was quantified in indicated strains. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. A one-way ANOVA combined with a Tukey test was performed to assess statistical significance. Underlying data for quantifications can be found in S1 Data. PTS, peroxisome targeting signal; RFP, red fluorescent protein. https://doi.org/10.1371/journal.pbio.3002508.g005 Crossings of a control strain expressing Ptc5-RFP-PTS and Pex3-GFP as a peroxisomal marker with arrayed libraries containing yeast deletion strains and hypomorphic mutants of essential genes were performed and sporulated to select for haploid cells containing both fluorescent proteins together with a genetic perturbation (Fig 5A) [41–43]. Inspection of haploid progeny by automated microscopy uncovered various genes affecting peroxisomal targeting of Ptc5-RFP-PTS (Figs 5B–5D and S8 and S2 Table). These include many expected genes involved in peroxisome biogenesis but also distinct genes, e.g., SOM1 and IMP1. Som1 was shown previously to be part of the IMP complex [44], which we could confirm (Figs 5C and S8).

Depletion of ERMES affects the distribution of dually targeted proteins Looking at the screen results, we found that an interesting gene whose deletion affected the co-localization between Ptc5 and peroxisomes was MDM10. Mdm10 is a critical factor for forming a contact site between mitochondria and the ER as part of the ER–mitochondria encounter structure (ERMES) complex (Fig 6A) [45]. The ERMES complex consists of the ER-anchored protein Mmm1 and 3 mitochondrial proteins (Mdm10, Mdm12, and Mdm34) [45]. The integral membrane protein Mdm10 has an additional function and controls import of mitochondrial ß-barrel proteins [46]. It was therefore unclear whether the effect of Mdm10 on Ptc5 localization was through the ERMES or by affecting mitochondrial protein import. PPT PowerPoint slide

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TIFF original image Download: Fig 6. ERMES complex regulates import of proteins into mitochondria and peroxisomes. (A) Subcellular localization of Ptc5-RFP-PTS (red), the peroxisomal membrane protein Ant1-YFP (green), and the mitochondrial inner membrane protein Tim50-CFP (blue) in control or Δmdm10 cells was analyzed with fluorescence microscopy. (B) Correlation between Ptc5-RFP-PTS signal and Ant1-YFP signal was quantified in indicated strains. (C) The truncated variant Ptc51-201-RFP lacking a PTS1 was expressed in indicated strains. Cleavage sites for MPP (filled arrow) and the IMP complex (blank arrow) are indicated in the scheme. Whole cell lysates were analyzed by SDS-PAGE and immunoblot. p: premature isoform, i: intermediate isoform, m: mature isoform. Concentrations of protein extracts were adapted to each other to focus on processing. (D) Cat2-RFP-HA-PTS (red) was co-expressed with Ant1-YFP (green) and Tim50-CFP (blue) in control or Δmdm10 cells. Subcellular localization was determined with fluorescence microscopy. White arrows denote peroxisomes overlapping mitochondria. (E) Fluorescence microscopic pictures of indicated strains expressing respective RFP fusion proteins (red) together with Ant1-YFP (green) and Tim50-CFP (blue). (F) Whole cell lysates of strains expressing the indicated fusion proteins were analyzed by SDS-PAGE and immunoblot. Concentrations of protein extracts were adapted to each other to focus on processing. (G) The number of Pxp2-positive foci per cell at mitochondria was quantified in indicated strains (left). Quantification of the fraction of mitochondrial Pxp2 foci overlapping Ant1-YFP (right). (H) Fluorescence microscopic picture of a strain expressing Pxp2-RFP-HA-PTS (magenta) together with Mmm1-mNeonGreen (green). White arrows indicate Pxp2-RFP-PTS foci overlapping Mmm1-mNeonGreen foci. Scale bars represent 5 μm. Quantifications are based on n = 3 experiments. Each color represents 1 experiment. Error bars represent SEM. P-values were calculated using a two-sided unpaired Student’s t test. For multiple comparisons, P-values were calculated with a one-way ANOVA combined with a Tukey test. Underlying data for quantifications can be found in S1 Data. ERMES, endoplasmic reticulum–mitochondria encounter structure; IMP, inner membrane peptidase; PTS, peroxisome targeting signal; RFP, red fluorescent protein. https://doi.org/10.1371/journal.pbio.3002508.g006 To address whether the observed phenotype is specific to Mdm10 or shared by other ERMES complex members, we repeated our assay in freshly made strains that have reduced levels of possible suppressor mutations. In all ERMES mutants targeting of Ptc5-RFP-PTS to peroxisomes was significantly reduced (Figs 6B and S9) with Δmdm10 displaying the strongest phenotype and Δmmm1 being least affected. To test the stage at which ERMES mutants affected dually targeted proteins, we first analyzed the initial steps of mitochondrial protein import. All mutants influenced mitochondrial import and preprotein processing as determined by testing a truncated variant of Ptc5-RFP (Fig 6C). Moreover, we observed an import defect for several bona fide mitochondrial proteins in Δmdm10 cells (S10A Fig) demonstrating that loss of ERMES complex altered translocation of mitochondrial proteins in a more general way. If compromised initial protein targeting of Ptc5-RFP-PTS to mitochondria was responsible for reduced peroxisomal targeting, mutants lacking components of the mitochondrial import machinery should exhibit a similar phenotype. As these were not identified in our screen, we tested if these were false negatives. We assessed Ptc5-RFP-PTS localization in Δtom22, Δtom70, and in a mutant lacking the mitofusin, Fzo1 that contains defective mitochondria similar to Δmdm10 (S10B Fig). None of these mutants showed mitochondrial retention of Ptc5-RFP-PTS, but preprotein processing defects were observed in FZO1 depleted cells (S10B and S10C Fig). In Δtom70 cells, a reduced but peroxisomal signal of Ptc5 was observed (S10B and S10C Fig), demonstrating that perturbation of the initial steps in mitochondrial import is not sufficient to block Ptc5 targeting to peroxisomes. The previously suggested binding partner of ERMES Pex11 [18] did not interfere with Ptc5 transit. The phenotype of ERMES mutants is therefore distinctive. To follow up on these results, we determined the localization of other proteins that contain competing targeting signals in Δmdm10 cells. Cat2-RFP-PTS behaved similarly to Ptc5-RFP-PTS and was retained in mitochondria while targeting to peroxisomes was reduced (Figs 6D and S11A). In contrast, the localization of Pxp2-RFP-PTS, Tes1-RFP-PTS, and Mss2-RFP-PTS displayed the opposite pattern of localization in Mdm10 depleted cells with reduced mitochondrial targeting but normal peroxisome targeting (Figs 6E and S11B). Proteolytic processing defects could not be observed for Pxp2, Tes1, and Mss2 suggesting a noncanonical MTS (Fig 6F). Quantification showed a reduction of mitochondrial foci containing Pxp2-RFP-PTS (Fig 6G). ERMES components could be directly involved in mitochondrial targeting as we found that Pxp2-RFP-PTS foci regularly (38% +/− 5%) overlapped with Mmm1-mNeonGreen foci (Fig 6H and S3 Table), a phenotype previously observed for a PerMit reporter, which also enhances contact when overexpressed [17]. Hence, contact forming proteins show a tendency to accumulate at ERMES sites. In aggregate, these data revealed ERMES as an important factor to determine the distribution of various dually targeted proteins. Interestingly, for different cargo either predominant peroxisomal targeting or mitochondrial targeting was observed.

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