Mitochondrial AAA‐ATPase Msp1 detects mislocalized tail‐anchored proteins through a dual‐recognition mechanism
2019; Springer Nature; Volume: 20; Issue: 4 Linguagem: Inglês
10.15252/embr.201846989
ISSN1469-3178
AutoresLanlan Li, Jing Zheng, Xi Wu, Hui Jiang,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle12 March 2019free access Transparent process Mitochondrial AAA-ATPase Msp1 detects mislocalized tail-anchored proteins through a dual-recognition mechanism Lanlan Li College of Life Sciences, Beijing Normal University, Beijing, China National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China Search for more papers by this author Jing Zheng National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Xi Wu Corresponding Author [email protected] orcid.org/0000-0002-9327-139X National Institute of Biological Sciences, Beijing, China Search for more papers by this author Hui Jiang Corresponding Author [email protected] orcid.org/0000-0002-0277-9711 National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China Search for more papers by this author Lanlan Li College of Life Sciences, Beijing Normal University, Beijing, China National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China Search for more papers by this author Jing Zheng National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China School of Life Sciences, Peking University, Beijing, China Search for more papers by this author Xi Wu Corresponding Author [email protected] orcid.org/0000-0002-9327-139X National Institute of Biological Sciences, Beijing, China Search for more papers by this author Hui Jiang Corresponding Author [email protected] orcid.org/0000-0002-0277-9711 National Institute of Biological Sciences, Beijing, China Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China Search for more papers by this author Author Information Lanlan Li1,2,3,‡, Jing Zheng2,3,4,‡, Xi Wu *,2,† and Hui Jiang *,2,3,5 1College of Life Sciences, Beijing Normal University, Beijing, China 2National Institute of Biological Sciences, Beijing, China 3Beijing Key Laboratory of Cell Biology for Animal Aging, Beijing, China 4School of Life Sciences, Peking University, Beijing, China 5Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China †Present address: BeiGene, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 58958215; E-mail: [email protected] *Corresponding author. Tel: +86 10 80723279; E-mail: [email protected] EMBO Rep (2019)20:e46989https://doi.org/10.15252/embr.201846989 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The conserved AAA-ATPase Msp1 is embedded in the outer mitochondrial membrane and removes mislocalized tail-anchored (TA) proteins upon dysfunction of the guided entry of tail-anchored (GET) pathway. It remains unclear how Msp1 recognizes its substrates. Here, we extensively characterize Msp1 and its substrates, including the mitochondrially targeted Pex15Δ30, and full-length Pex15, which mislocalizes to mitochondria upon dysfunction of Pex19 but not the GET pathway. Moreover, we identify two new substrates, Frt1 and Ysy6. Our results suggest that mislocalized TA proteins expose hydrophobic surfaces in the cytoplasm and are recognized by Msp1 through conserved hydrophobic residues. Introducing a hydrophobic patch into mitochondrial TA proteins transforms them into Msp1 substrates. In addition, Pex15Δ30 and Frt1 contain basic inter-membrane space (IMS) residues critical for their mitochondrial mistargeting. Remarkably, Msp1 recognizes this feature through the acidic D12 residue in its IMS domain. This dual-recognition mechanism involving interactions at the cytoplasmic and IMS domains of Msp1 and substrates greatly facilitates substrate recognition and is required by Msp1 to safeguard mitochondrial functions. Synopsis Outer mitochondrial membrane AAA-ATPase Msp1 removes mislocalized tail-anchored proteins to safeguard mitochondrial function. Msp1 detects substrates through a dual recognition mechanism involving interactions in the cytoplasm and the intermembrane space. The Msp1 N-domain contains conserved residues critical for substrate recognition and removal. Hydrophobic residues in the cytoplasmic region of the Msp1 N-domain interact with exposed hydrophobic surfaces of mislocalized tail-anchored proteins. In the intermembrane space, some mislocalized tail-anchored proteins contain positively-charged residues critical for their mistargeting and are recognized by the Msp1 D12 residue through electrostatic interactions. Introduction Mitochondria are essential organelles that play pivotal roles in energy supply, metabolism, and signaling processes such as cell death. Mitochondrial proteostasis is critical for mitochondrial fitness and is maintained by AAA-proteases 1, 2, the ubiquitin-Cdc48-proteasome pathway 3-5, and an AAA-ATPase Msp1 embedded in outer mitochondrial membrane (OMM) that clears mislocalized tail-anchored (TA) proteins 6, 7. TA proteins are post-translationally targeted to the ER, peroxisome, and mitochondria 8. The best understood TA protein targeting pathway is the guided entry of tail-anchored (GET) pathway: Newly synthesized TA protein is recognized at the transmembrane (TM) segment by the chaperone Sgt2, then delivered to the ATPase Get3, and finally transferred to the Get1/2 insertase complex at ER membrane 9, 10. In get mutant cells, many GET-dependent TA proteins accumulate as aggregates in the cytoplasm, and a subset of them are mistargeted to mitochondria 11. OMM contains important TA proteins, including the fission receptor Fis1 12, the ER-mitochondria encounter structure component Gem1 13, and subunits of the TOM import complex including Tom5, Tom6, Tom7, and Tom22 14. It remains unclear that how TA proteins are targeted to mitochondria as none of the known mitochondrial import machineries is required for their biogenesis 15, 16. Msp1 is an evolutionarily conserved AAA-ATPase (ATAD1 in human) that dually localizes to mitochondria and peroxisome. It removes TA proteins mistargeted to mitochondria in get mutant cells to safeguard mitochondrial function. Synthetic mutations of MSP1 and GET genes cause the accumulation of mistargeted TA proteins on mitochondria, resulting in severe mitochondrial defects and poor respiratory growth 6, 7. Peroxisomal Msp1 clears excessive TA protein Pex15 that fails to assemble into complex with its binding partner Pex3 17. ATAD1 knockout mice and patients carry ATAD1 mutations show severe mitochondrial damages 6, 18. It is interesting and essential to understand how Msp1 detects and distinguishes its substrates from mitochondrial TA proteins. Msp1 belongs to the meiotic clade of AAA proteins that contains an N-domain followed by an AAA-ATPase domain 19. Other members of this clade include Vps4, Spastin, Katanin, and Fidgetin. Studies of these four enzymes suggest common features that they exist as monomers/dimers, directly bind substrates through the N-terminal microtubule interacting and trafficking (MIT) domain, and assemble into hexamer upon substrate engagement 20. Msp1 alone is sufficient to dislocate TA protein from liposome as shown in an in vitro assay 21, indicating Msp1 can directly recognize substrates. But as the only transmembrane protein in this clade, Msp1 possesses a different N-domain that contains a TM segment and lacks homology to those of other members 19. Furthermore, epichromosomally expressed Msp1 N-domain mutants can rescue msp1Δ cells 21, arguing against a role of Msp1 N-domain in substrate recognition. Msp1 also interacts with Cis1, a stress-responsive protein that facilitates the Msp1-dependent removal of mitochondrial precursor proteins clogged in the TOM complex 22. Whether Cis1 is involved in the removal of mistargeted TA proteins remains unclear. In this study, we combine genetic, biochemical, and imaging approaches to address the molecular mechanisms of substrate recognition by Msp1. Results Identification of Msp1 N-domain residues critical for GFP-Pex15Δ30 binding Msp1 function was determined by monitoring the degradation of its model substrate GFP-Pex15Δ30, in which the last 30 amino acids of Pex15 were truncated to make it constitutively targeted to mitochondria 7. Cis1 knockout did not affect GFP-Pex15Δ30 degradation (Appendix Fig S1). We thus focused on Msp1 itself. Msp1 consists of an N-domain of 98 amino acids, and a C-terminal AAA-domain highly analogous to other AAA-ATPases (Fig 1A). We rescued msp1Δ cells with wild-type (WT) Msp1 or Msp1TOM70(N), in which the N-terminal 32 residues of Msp1 were replaced with those of Tom70 (Fig EV1A). When expressed by epichromosomal vector under the control of Msp1 promoter, Msp1TOM70(N) restored GFP-Pex15Δ30 degradation and rescued the growth defect of get3Δ msp1Δ cells under respiratory growth condition (SCEG; Fig EV1B and C), consistent with previous results 21. In contrast, when expressed by knockin, Msp1Tom70(N) did not rescue these defects (Fig EV1B and C). Epichromosomal expression produced three times more proteins than the knockin method (Fig EV1D), suggesting that Msp1 N-domain contains key residues facilitating substrate degradation, and the loss of these residues requires compensation by Msp1 overexpression. Furthermore, epichromosomal expression resulted in heterogeneous expression (Fig EV1E). We thus utilized the knockin method to analyze Msp1 N-domain mutants. Figure 1. Identification of residues in Msp1 N-domain essential for GFP-Pex15Δ30 binding Alignment of Msp1 N-domain sequence (amino acids 1–98) with those of its orthologs. Residues marked by asterisks were tested for their roles in GFP-Pex15Δ30 degradation. The critical residues are highlighted in red. Degradation of GFP-Pex15Δ30 in strains expressing WT or mutant forms of Msp1-FLAG. Cells were grown in synthetic glucose media (SCD) to log phase, treated with cycloheximide (CHX) to stop protein synthesis, and collected at the indicated time points. L122, 123D mutation disrupts Msp1 hexamer assembly and thus served as a control for Msp1 loss of function. Anti-Por1 blots were shown as the loading controls. Hexamer assembly by WT and mutant forms of Msp1-FLAG. Mitochondria-enriched fraction was isolated from the indicated knockin strains, lysed with 1% digitonin. Lysates (10 μg) were analyzed by blue-native (BN) PAGE or SDS–PAGE. Interaction of GFP-Pex15Δ30 with WT and mutant forms of Msp1-FLAG. Mitochondria were isolated, lysed with 1% digitonin, and subject to anti-FLAG immunoprecipitation (IP). Mitochondrial extracts (10 μg) and immunoprecipitates (100 μl in total, load 1 μl for FLAG and 10 μl for GFP blot) were analyzed by Western blot. The intensity ratio of GFP-Pex15Δ30/Msp1-FLAG was calculated using ImageJ software and normalized to the value of positive control (GFP-Pex15∆30/Msp1E193Q-FLAG). The positions of conserved critical residues for substrate binding in the monomeric (PDB ID: 5W0T) and hexameric structures of Msp1 21. Three subunits of the hexamer were removed to visualize pore loop 1 (yellow). The AAA-domain is in light gray and part of the N-domain (amino acids 58–98) is in light green. Critical residues are highlighted in red. OMM: outer mitochondrial membrane. Data information: In this figure, GFP-Pex15Δ30 was expressed from a centromeric plasmid under the control of TEF1 promoter, and WT and mutant forms of Msp1-FLAG were expressed from the endogenous chromosomal locus. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. N-domain is essential for Msp1 function under endogenous expression level (associated with Fig 1) Domain structure of Msp1 and Msp1Tom70(N). Msp1Tom70(N) was constructed by replacing the first 32 amino acids of Msp1 with that of Tom70. Msp1Tom70(N)-GFP, when expressed at endogenous level by the knockin method, failed to restore mRFP-HA-Pex15Δ30 degradation. mRFP-6xHA-tagged Pex15Δ30 was expressed from a centromeric plasmid under the control of TEF1 promoter. The gene cassettes expressing WT or mutant forms of Msp1 under the control of MSP1 endogenous promoter were either integrated at the endogenous locus (knockin) or cloned into a centromeric plasmid (epichromosome). The indicated strains were grown in synthetic glucose media to log phase and then treated with CHX and collected at the indicated time points. Msp1Tom70(N)-GFP, when expressed at endogenous level by the knockin method, failed to rescue the growth defect of get3Δ msp1Δ cells. The indicated strains constructed similarly as in (B) were grown in glucose media to log phase and then spotted on SCD or SCEG plates in a 10-fold serial dilution, and then incubated for 2–5 days. The relative protein level of chromosomally and epichromosomally expressed Msp1TOM70(N)-GFP. The intensity ratios of Msp1/Por1 were calculated using ImageJ software, and the value in WT was set to 1. Heterogeneous levels of Msp1TOM70(N)-GFP expressed from a centromeric plasmid. Z projections and DIC images are shown. Scale bar represents 1 μm. Download figure Download PowerPoint Alignment of the N-domain sequences of Msp1 and its orthologs revealed conserved residues (Fig 1A). We performed mutagenesis scan of 39 residues within and flanking the TM domain and in the conserved C-terminal region (marked by asterisks in Fig 1A). The L122, 123D mutant disrupts Msp1 hexamer assembly 21 and thus served as a control for Msp1 loss of function. We identified 11 residues whose mutations did not affect Msp1 localization (Fig EV2A) and stability (Fig 1B) but impaired GFP-Pex15Δ30 degradation (Figs 1B and EV2B). The untagged Msp1 V81A and I93A mutants also impaired substrate degradation (Fig EV2C). We further examined whether Msp1 stability and folding were affected by the mutants. The thermostability of endogenous Msp1 was not affected by V81A and I93A mutations (Appendix Fig S2A–D). We then generated recombinant proteins of Msp1 cytoplasmic domain. WT Msp1 and its V81A, I86A, I93A, and G95A mutants had similar sensitivity to limited trypsin digestion (Appendix Fig S2E). These results indicate the N-domain mutants did not alter Msp1 structure. These residues are highlighted in red (Fig 1A) and most of them are evolutionarily conserved. Notably, D12 is lost in the Msp1TOM70(N) mutant. Click here to expand this figure. Figure EV2. The localization of Msp1 N-domain mutants and their effects on GFP-Pex15Δ30 degradation (associated with Fig 1) The normal localization of Msp1 N-domain mutants. Z projections and DIC images are shown. Mitochondrial Msp1-GFP colocalizes with mtBFP. Peroxisomal Msp1 appears as extra-mitochondrial dots. Scale bar represents 1 μm. Degradation of GFP-Pex15Δ30 in WT and Msp1 N-domain mutant cells. Mutants displaying defective degradation are highlighted in red. Degradation of GFP-Pex15Δ30 in untagged Msp1 N-domain mutant cells. msp1E193Q-FLAG strain is used as a control for Msp1 loss of function. Data information: In this figure, WT and mutant forms of Msp1-GFP and Msp1-FLAG were expressed from the endogenous chromosomal locus, and GFP-Pex15Δ30 was expressed from a centromeric plasmid under the control of TEF1 promoter. Download figure Download PowerPoint The E193Q walker B mutant of Msp1 has normal ATP binding but loses ATP hydrolysis activity. It forms a hexamer and constitutively binds substrates and thus functions as a substrate-trap mutant 6, 7, 21. When assayed by blue-native gel, Msp1-FLAG existed as monomers and Msp1E193Q-FLAG as hexamers (Fig 1C, lanes 2 and 3), a typical feature of meiotic clade of AAA-ATPases 20. Msp1L122,123D,E193Q served as a control to disrupt hexamer assembly. Most mutations did not affect the oligomerization of Msp1E193Q-FLAG except Y72A, which caused a slight increase of monomeric forms (Fig 1C, lane 7). We then analyzed substrate binding with Msp1. As expected, Msp1E193Q-FLAG pulled down GFP-Pex15Δ30, whereas Msp1-FLAG did not (Fig 1D, lanes 2 and 3). Nearly, all the mutations except G94A impaired GFP-Pex15Δ30 pulldown by Msp1E193Q-FLAG (Fig 1D, lanes 5–15). Therefore, most of the residues are required for substrate binding. We highlighted all the conserved residues critical for substrate binding on Msp1 monomeric (PDB ID: 5W0T) and hexameric structures 21 (Fig 1E): D12 is a negatively charged residue in the inter-membrane space (IMS) and the other residues are in the cytoplasm. Notably, most cytoplasmic critical residues are in the folded region and are hydrophobic residues, except E73 and G95. Y72, V81, I86, I93, and L96 are in contact with the AAA-domain (light gray), most possibly to avoid exposing hydrophobic surfaces to the cytoplasm. Hydrophobic residues L69 and Y72, especially the latter, are close to pore loop 1, which is critical for substrate translocation into the pore 21. Characterization of Msp1 and GFP-Pex15Δ30 interaction by in vivo site-specific photo-crosslinking Msp1 N-domain may interact with substrates through the IMS and cytoplasmic regions because critical residues are present in both regions. We thus characterized the interaction between Msp1E193Q-FLAG and GFP-3xHA-Pex15Δ30 by the in vivo site-specific photo-crosslinking method 23, 24. Photo-activatable amino acid p-benzoyl-L-phenylalanine (BPA) was introduced at a position specified by an amber codon in the target protein. Upon UV irradiation of living cells, BPA photo-crosslinks with direct interacting proteins. By sampling multiple positions, the interaction surface consisting of residues positive for BPA photo-crosslinking can be mapped. We and others have utilized the method to characterize mitochondrial complexes 25-27. Substrate-trap mutants of AAA-ATPase can engage substrates into the central pore formed by the ATPase hexamer 28, which contains three conserved pore loops critical for substrate translocation (Fig 2A) 21. BPA incorporated at Msp1E193Q pore loops crosslinked with GFP-3xHA-Pex15Δ30 (Fig 2B, crosslinked residues are color-labeled in Fig 2A), indicating Msp1 AAA-domain works in a similar manner as other AAA-ATPases. Figure 2. Characterization of GFP-3xHA-Pex15Δ30 and Msp1E193Q-FLAG interaction by in vivo site-specific photo-crosslinking A. A hexameric model of Msp1 with pore loop residues positive for crosslinking highlighted by colors. B, C. In vivo photo-crosslinking of BPA incorporated in pore loops (B) and N-domain (C) of Msp1E193Q-FLAG with GFP-3xHA-Pex15Δ30. D. In vivo photo-crosslinking of BPA incorporated in GFP-3xHA-Pex15Δ30 (amino acids 260–352) with Msp1E193Q-FLAG. Experiments in (B–D) were performed as described in Materials and Methods. Immunoprecipitates (100 μl in total, load 1 μl for FLAG, and 40–50 μl for HA blot) were analyzed by Western blot. IMS, inter-membrane space. E. Cartoon summary of photo-crosslinking results. The sites showing strong (filled red) and weak or no (red circle) crosslinking are highlighted in the topology model. The boxed region highlights the position of N-domain (amino acids 50–98, shown in green) in the hexameric model of Msp1. N-domain residues positive for crosslinking (red), hydrophobic residues (L69 and Y72, blue) critical for substrate binding, and pore loop 1 (yellow) are highlighted. Download figure Download PowerPoint We then incorporated BPA into Msp1E193Q N-domain (Fig 2C). We excluded most critical residues of Msp1 N-domain from BPA incorporation because incorporating BPA into these residue positions is similar to alanine mutation and may disrupt substrate interaction and cause false-negative results. We also incorporated BPA into residues 260–352 of Pex15Δ30 (Fig 2D), which contains all the critical elements for recognition by Msp1 (see Fig 3 for evidence). As summarized in Fig 2E, two features and indications can be obtained: (i) Direct interaction between Msp1 N-domain and Pex15Δ30 extensively occurs in their IMS, TM, and cytoplasmic regions. (ii) The folded region of Msp1 N-domain (amino acids 50–98, shown in green in the structural models) lines along the surface of Msp1 hexamer to positions near pore loop 1 and has direct interactions with Pex15Δ30 (crosslinked residues shown in red in the structural models). This spatial organization may facilitate substrate transfer from N-domain into the central pore. Figure 3. The IMS tail charge and cytoplasmic hydrophobic patch of Pex15Δ30 are required for its recognition and removal by Msp1 Degradation of GFP-Pex15Δ30 by Msp1 D12 mutants. Schematic illustration of the Outer mitochondrial membrane Targeting Sequences (OTSs) of GFP-Pex15Δ30 and mitochondrial TA proteins. The GRAVY (Grand Average of Hydropathicity) values of TM domain (highlighted in orange) and the charges of the flanking sequences are shown on the right. Degradation of GFP-Pex15Δ30 and its chimeric variants by Msp1. Interaction of GFP-Pex15Δ30 and its chimeric variants with Msp1. Experiments were performed as in Fig 1D. Hydrophobicity plot of Pex15Δ30 using the Kyte–Doolittle scale. The hydrophobic patch and the TM domain are highlighted in green and orange, respectively. Truncated forms of Pex15Δ30 are shown below. Degradation of GFP-Pex15Δ30 and its truncation mutants. Interaction of GFP-Pex15Δ30 and its truncation mutants with Msp1. Cells were analyzed as in (D). Data information: In this figure, GFP-Pex15Δ30 and its mutants were expressed from a centromeric plasmid under the control of TEF1 promoter, and WT and mutant forms of Msp1-FLAG were expressed from the endogenous chromosomal locus. Download figure Download PowerPoint Positively charged residues in IMS tail of GFP-Pex15Δ30 correlate with its recognition and removal by Msp1 The negatively charged D12 residue of Msp1 is critical for substrate binding (Fig 1D). Msp1 D12E mutation did not affect, whereas D12A and D12K mutations impaired substrate degradation (Fig 3A), indicating the negative charge of D12 is critical for its function. D12 may interact with the IMS tail of substrates through electrostatic interactions considering the presence of three positively charged K residues in Pex15Δ30 IMS tail (Fig 3B). We mutate these K residues, but even mutating one K to H mistargeted GFP-Pex15Δ30 to the cytoplasm (Fig EV3A). This result is consistent with previous observations that positively charged residues in IMS tail are critical for the mitochondrial targeting of many TA proteins 8, 29, 30. Click here to expand this figure. Figure EV3. Localization of WT or mutant forms of GFP-Pex15Δ30, GFP-Gem1, and GFP-Fis1 (associated with Figs 3 and 4) Localization of GFP-Pex15Δ30 carrying the indicated Outer mitochondrial membrane Targeting Sequence (OTS). Mutations in the IMS tail are highlighted in red. Localization of GFP-Pex15Δ30 carrying indicated OTS in msp1Δ cells. Localization of GFP-Pex15Δ30 truncation mutants in msp1E193Q cells. Localization of GFP-Pex15Δ30 hydrophobic patch mutants in msp1E193Q cells. Localization of GFP-Gem1 and its insertion mutants in msp1E193Q cells. Localization of GFP-Fis1 and its insertion mutants in msp1E193Q cells. Data information: In this figure, GFP-tagged TA proteins and their mutants were expressed from a centromeric plasmid under the control of TEF1 promoter. Z projections and DIC images are shown. Scale bars represent 1 μm. Download figure Download PowerPoint Outer mitochondrial membrane TA proteins Tom5, Gem1, and Fis1 carry IMS sequences rich in positively charged residues, but Tom6 does not (Fig 3B). We thus replaced the OMM targeting sequence (OTS) of GFP-Pex15Δ30 with that of OMM TA proteins. The chimeric proteins correctly localized to mitochondria (Fig EV3B). OTSs with positively charged residues (Tom5, Gem1, and Fis1) supported the degradation of chimeric proteins by Msp1 (Fig 3C). Similar to GFP-Pex15Δ30, the degradation was impaired by the Msp1D12T mutation (Fig 3C). In contrast, the chimeric protein with Tom6 OTS was stable (Fig 3C). Consistent with the degradation results, OTSs of Tom5, Gem1, and Fis1 but not Tom6 supported substrate interaction with Msp1 (Fig 3D). We also tried to reduce positive charges in Tom5, Gem1, and Fis1 IMS tails. But again all the mutants tested were mislocalized (Fig EV3A). Taken together, these results support the idea that positively charged residues in substrate IMS tail facilitate their interaction with Msp1, very likely through electrostatic interactions with Msp1 D12 residue. A hydrophobic patch in GFP-Pex15Δ30 cytoplasmic domain is essential for its recognition and removal by Msp1 The enrichment of hydrophobic critical residues in Msp1 N-domain suggests hydrophobic interactions might occur between Msp1 and substrate cytoplasmic domains. Hydrophobicity analysis revealed a hydrophobic patch near Pex15 TM segment (Fig 3E). The hydrophobic patch is rich in residue positions showing crosslinking with Msp1 (Fig 2E). Serial truncations showed that a minimum sequence (Δ1–311) consisting of the hydrophobic patch, TM segment, and IMS tail is sufficient for interaction with and removal by Msp1 (Fig 3F and G). Further deletion of the hydrophobic patch (Δ1–324) rendered the protein stable and unrecognized by Msp1 (Fig 3F and G). Deleting the hydrophobic patch alone (Δpatch) abolished the interaction between GFP-Pex15Δ30 and Msp1 (Fig 3G), and caused GFP-Pex15Δ30 degradation by an Msp1-independent mechanism (Fig 3F). All the GFP-Pex15Δ30 truncation mutants correctly localized to mitochondria (Fig EV3C). These results demonstrate that the hydrophobic patch of GFP-Pex15Δ30 is essential for its recognition and removal by Msp1. In the patch, I313, F320, and L324 likely form a hydrophobic core, with potential contribution from flanking L316 and A317 (Fig 4A). To determine whether this hydrophobic core is required for GFP-Pex15Δ30 recognition by Msp1, we mutated three residues into non-hydrophobic ones (I313S, F320S, and L324G). Surprisingly, this mutant form mislocalized to the cytoplasm (Fig EV3D), suggesting hydrophobic residues in the patch are critical for mitochondrial targeting. We then mutated these residues to A (Fig 4B, mut1), which is of medium hydrophobicity and correctly localized to mitochondria (Fig EV3D). GFP-Pex15Δ30-mut1 degradation was partially delayed in msp1E193Q cells and mainly degraded by an Msp1-independent mechanism (Fig 4B), similar as the degradation of the Δpatch mutant (Fig 3F). We screened a panel of mutants and found that temperature-sensitive inactivation of the ATPase subunits of proteasome Cim3 delayed GFP-Pex15Δ30-mut1 degradation. Inactivating Cim3 in msp1Δ cells efficiently blocked GFP-Pex15Δ30-mut1 degradation (Fig 4C). GFP-Pex15Δ30-mut1 accumulated on mitochondria in Msp1 and Cim3 mutant strains (Fig 4D), allowing us to examine its interaction with Msp1. In comparison with GFP-Pex15Δ30, GFP-Pex15Δ30-mut1 had significantly weaker interaction with Msp1E193Q-FLAG (Fig 4E, lane 3 vs. 6) and the weak interaction was not enhanced by inactivating Cim3 (Fig 4E, lane 6 vs. 9). Collectively, these results suggest that the hydrophobic core in the patch of GFP-Pex15Δ30 is crucial for its recognition by Msp1. Figure 4. Patch hydrophobicity of Pex15Δ30 is critical for its interaction with Msp1 and insertion of the patch into mitochondrial TA proteins Fis1 and Gem1 transforms them into Msp1 substrates Helical wheel plot of the hydrophobic patch of Pex15 (amino acids 313–324). Residues are color-coded according to the hydrophobicity scores. Degradation of GFP-Pex15Δ30 with WT or mutant hydrophobic patch in the indicated strains. Shown on the left are helical wheel plots of WT and mutant (mut1) hydrophobic patches of Pex15. Residue hydrophobicity is color-coded as in (A). The mut1 form carries I313A, L316A, F320A, and L324A mutations. Degradation of GFP-Pex15Δ30-mut1 in the indicated mutants. Cim3-1 is a temperature-sensitive mutant of Cim3. Cells were cultured at 37°C for 1 h to inactivate Cim3 and then treated with CHX. Localization of GFP-Pex15Δ30-mut1 to mitochondria in the indicated strains. Cells were cultured at 37°C for 1 h before imaging. Scale bar represents 1 μm. Interaction of WT and mutant GFP-Pex15Δ30 with Msp1- and Msp1E193Q-FLAG. Cells were cultured at 37°C for 1 h before mitochondria purification. Mitochondrial extracts were subject to anti-FLAG IP and analyzed as in Fig 1D. Insertion of Pex15 hydrophobic patch but not its hydrophilic mutants transforms Gem1 into Msp1 substrate. The patch was inserted between V628 and D629 of Gem1 (before Gem1 OTS) as shown in the upper panel. The mut2 form carries I313N, A317R, F320R, and L324Q mutations. The mut3 form carries I313N, L316Q, F320R, L324Q mutations. Interaction of GFP-Gem1 and its insertion mutants with Msp1- and Msp1E193Q-FLAG. Mitochondrial extracts were subject to anti-FLAG IP and analyzed as in Fig 1D. Insertion of Pex15 hydrophobic patch before Fis1 OTS but not at the N-terminus transforms Fis1 into Msp1 substrate. The insertion was placed between Q125 and K126 of Fis1 (before Fis1 OTS) or at the N-terminus. The mut4 form carries I313N, F320R, L324Q mutations. Data information: In this figure, GFP-tagged Pex15Δ30, Gem1, Fis1, and their mutants were express
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