Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane

Short Article

Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane Graphical Abstract

Authors Lars Ellenrieder, Martin P. Dieterle, Kim Nguyen Doan, ..., Marı´a Luisa Campo, Nikolaus Pfanner, Thomas Becker

Correspondence nikolaus.pfanner@ biochemie.uni-freiburg.de (N.P.), thomas.becker@ biochemie.uni-freiburg.de (T.B.)

In Brief Ellenrieder et al. report that the major metabolite channel porin of the mitochondrial outer membrane promotes the import of carrier proteins to the mitochondrial inner membrane. Porin binds carrier precursors in the intermembrane space and recruits the carrier translocase of the inner membrane to facilitate transfer of the precursor proteins.

Highlights d

Metabolite channel of mitochondrial outer membrane promotes protein import

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Porin interacts with carrier precursors accumulated in the intermembrane space

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Porin recruits the carrier translocase of the inner mitochondrial membrane

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Dual role of porin as metabolite channel and as coupling factor in protein import

Ellenrieder et al., 2019, Molecular Cell 73, 1–10 March 7, 2019 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.molcel.2018.12.014

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Molecular Cell

Short Article Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane Lars Ellenrieder,1,2 Martin P. Dieterle,1 Kim Nguyen Doan,1,2 Christoph U. Ma˚rtensson,1,2 Alessia Floerchinger,1 Marı´a Luisa Campo,3 Nikolaus Pfanner,1,4,5,* and Thomas Becker1,4,* 1Institute

of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany of Biology, University of Freiburg, 79104 Freiburg, Germany 3Departamento de Bioquı´mica y Biologı´a Molecular y Gene ´ tica, Universidad de Extremadura, 10003 Ca´ceres, Spain 4CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany 5Lead Contact *Correspondence: [email protected] (N.P.), [email protected] (T.B.) https://doi.org/10.1016/j.molcel.2018.12.014 2Faculty

SUMMARY

The mitochondrial inner membrane harbors a large number of metabolite carriers. The precursors of carrier proteins are synthesized in the cytosol and imported into mitochondria by the translocase of the outer membrane (TOM) and the carrier translocase of the inner membrane (TIM22). Molecular chaperones in the cytosol and intermembrane space bind to the hydrophobic precursors to prevent their aggregation. We report that the major metabolite channel of the outer membrane, termed porin or voltage-dependent anion channel (VDAC), promotes efficient import of carrier precursors. Porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring an efficient transfer of the precursors to the inner membrane translocase. Porin channel mutants impaired in metabolite transport are not disturbed in carrier import into mitochondria. We conclude that porin serves distinct functions as outer membrane channel for metabolites and as coupling factor for protein translocation into the inner membrane.

INTRODUCTION Mitochondria play a central role in cellular metabolism and ATP production. The two mitochondrial membranes have to transport a large number of small molecules like metabolites and ions to integrate mitochondria into the cellular metabolism. The mitochondrial outer membrane contains several channel-forming €ger et al., 2017), including the major metabolite proteins (Kru transporter porin, also termed voltage-dependent anion channel (VDAC) (Colombini, 2012; Messina et al., 2012; Campo et al., 2017). The mitochondrial inner membrane harbors a large number of different metabolite carrier proteins to ensure specific transport of small molecules (Palmieri and Pierri, 2010).

Mitochondrial proteins encoded by nuclear genes are synthesized as precursors on cytosolic ribosomes and recognized by receptors on the mitochondrial surface. Proteins destined for internal mitochondrial compartments cross the outer membrane via the translocase of the mitochondrial outer membrane (TOM). Two main protein translocases of the inner mitochondrial membrane (TIM) sort precursor proteins into and across the inner membrane. The presequence translocase (TIM23) transports preproteins that carry characteristic amino-terminal targeting signals (presequences) into the inner membrane and matrix. The carrier translocase (TIM22) mediates membrane integration of hydrophobic precursor proteins such as metabolite carriers, which contain internal targeting signals but no presequences (de Marcos-Lousa et al., 2006; Neupert and Herrmann, 2007; Endo and Yamano, 2009; Wiedemann and Pfanner, 2017). The import of carrier proteins into mitochondria is a multistep process (Figure 1A) (de Marcos-Lousa et al., 2006; Wiedemann and Pfanner, 2017). Cytosolic chaperones bind to the hydrophobic precursors and deliver them to the receptor Tom70 of the TOM complex (Wiedemann et al., 2001; Young et al., 2003). After passage through the Tom40 channel, the precursors bind to hexameric small TIM chaperone complexes that prevent aggregation of the precursors in the intermembrane space (Truscott et al., 2002; Webb et al., 2006; Shiota et al., 2015). The small TIM chaperones dock onto the TIM22 complex (Endres et al., 1999; Lionaki et al., 2008). The TIM22 complex is a multisubunit machinery with the channel-forming protein Tim22 as core subunit (Rehling et al., 2003; Peixoto et al., 2007). Tim22 mediates membrane insertion of the precursors in a membrane potential (Dc)-dependent manner (Figure 1A). The translocase contains additional subunits that are involved in assembly and stability of the complex (Tim29 and acylglycerol kinase in metazoa; Tim54, Tim18, and Sdh3 in fungi); Tim54, Tim29, and acylglycerol kinase are exposed to the intermembrane space and may function as docking sites (de Marcos-Lousa et al., 2006; Wagner et al., 2008; Gebert et al., 2011; Kang et al., 2018). The mechanisms that coordinate outer and inner membrane transport steps of carrier precursors are poorly understood. Here, we report the unexpected finding that the major metabolite channel porin (Por1) of the outer membrane promotes the Molecular Cell 73, 1–10, March 7, 2019 ª 2018 Elsevier Inc. 1

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Figure 1. Porin Is Linked to the Carrier Import Pathway of Mitochondria (A) Hypothetical model of the import stages of carrier proteins (Ryan et al., 1999; Rehling et al., 2003). The precursors are synthesized in the cytosol and are bound to molecular chaperones (stage I). Binding to the receptor Tom70 on the outer membrane (OM) represents stage II. Subsequently, the precursor is translocated through the TOM channel to the small TIM chaperones in the intermembrane space (IMS) (stage III). In the presence of a membrane potential (Dc), the precursor is inserted by the TIM22 complex (stage IV) and released into the inner membrane (IM) (stage V). (B) Serial dilutions of the indicated yeast strains were grown on full medium containing a fermentable carbon source (glucose-YPD [yeast extract, bacto-peptone, and glucose]) at the indicated temperatures. (C) Wild-type (WT) and por1D cell extracts were analyzed by SDS-PAGE and immunodetection. Cells were grown on YPD at 30
C and shifted to 37
C for 8 h (lanes 5–8) or left at 30
C (lanes 1–4). Protein, total protein amounts of 25–50 mg. (D) Mitochondria (lanes 1–12) or total cell extracts (lanes 13–16) from WT and a POR1-overexpressing (Por1[) yeast strain were analyzed by SDS-PAGE and immunodetection. Protein, total protein amounts of 10, 20, and 40 mg (lanes 1–12) or 25–50 mg (lanes 13–16). See also Figure S1.

import of carrier proteins into the inner membrane. Mutants with a double deletion of POR1/TOM70 or POR1/TIM18 show synthetic growth defects. Experimental dissection of the carrier import pathway reveals that porin facilitates the transfer of carrier precursors to the TIM22 complex. Porin tethers the TIM22 complex to the outer membrane to spatially coordinate inner and outer membrane transport steps. Mutants that inhibit metabolite transport do not affect porin’s function in the carrier import, pointing to two distinct roles of porin. We propose that the role of porin in biogenesis of carrier proteins represents a coupling mechanism between mitochondrial outer and inner membranes. RESULTS Genetic Link of Porin to the Carrier Import Pathway To identify novel components involved in the import of carrier proteins, we searched for genetic interaction partners of the non-essential subunits Tom70 and Tim18 of the carrier import pathway. Single deletion of either TOM70 or TIM18 mainly affected growth of yeast cells at 37
C (Figure 1B), whereas parallel disruption of POR1 and either TOM70 or TIM18 led to severe

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synthetic growth defects at 23
C and 30
C (Figure 1B). A double deletion of POR1 and SDH3, which codes for Sdh3 that is located in the TIM22 complex as well as the respiratory complex II (Gebert et al., 2011), also led to a strong synthetic growth defect (Figure S1A). The single deletion strain por1D is not viable at 37
C but can grow at lower temperature (Figure 1B) (BlachlyDyson et al., 1997). In yeast, POR1 encodes the highly abundant functional isoform Por1, whereas Por2 is present only in tiny amounts (four orders of magnitude below Por1) (Blachly-Dyson et al., 1997; Lee et al., 1998; Morgenstern et al., 2017). We asked whether Por1 is important for maintaining normal steady-state levels of metabolite carriers. The levels of carrier proteins analyzed, including ADP/ATP carrier (AAC), phosphate carrier (PiC), and oxaloacetate carrier (Oac1), were reduced in por1D cells at 30
C and more strongly diminished upon shifting the cells to non-permissive growth temperature, whereas the levels of Tom proteins remained stable (Figure 1C). The levels of carrier proteins were further decreased in a por1D tom70D double deletion mutant (Figure S1B). Upon overexpression of Por1, the levels of carrier proteins such as AAC, Oac1, and dicarboxylate carrier (DIC) were moderately increased; the levels of Tom and Tim proteins and various mitochondrial control proteins were

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

(legend on next page)

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Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

not increased (Figure 1D). These in vivo results suggest that outer membrane porin is linked to the biogenesis of inner membrane carrier proteins. Porin Is Required for the Efficient Import of Carrier Precursors into Mitochondria To study whether porin is required for the translocation of carrier proteins into mitochondria, radiolabeled carrier precursors were imported into isolated por1D mitochondria, and the import reaction was analyzed by blue native electrophoresis (Gebert et al., 2011). The import of carrier proteins was impaired in the absence of Por1 (Figure 2A). We performed several controls to analyze the specificity of the import defects. (1) Re-expression of POR1 in por1D yeast cells restored the carrier import (Figure 2B). Mitochondria isolated form three different Saccharomyces cerevisiae por1D strains showed comparable carrier import defects (Figure 2A, legend). (2) The membrane potential was only mildly reduced in por1D mitochondria (Figure S2A). The import of presequence-containing proteins via the TOM-TIM23 pathway was not inhibited in por1D mitochondria, as shown with the matrixtargeted model preproteins Su9-DHFR and F1-ATPase subunit b (F1b) as well as the inner membrane-targeted precursor of cytochrome c1 (Figures 2C and S2B). The efficient import of preproteins like F1b that is particularly sensitive to a decrease of Dc (Schendzielorz et al., 2017) excludes that the mild reduction of Dc was responsible for the carrier import defect of por1D mitochondria. (3) Addition of reduced glutathione (GSH) did not restore the carrier import in por1D mitochondria (Figure S2C), indicating that the import defect was not caused by the enhanced oxidized state of the por1D intermembrane space (Kojer et al., 2012). (4) The mitochondrial signature phospholipid cardiolipin is involved in assembly and function of carriers like the ADP/ATP carrier (Jiang et al., 2000). Comparison of the phospholipids of wild-type and por1D mitochondria under our growth conditions did not reveal decreased levels of cardiolipin (Figure S2D). (5) The import of outer membrane proteins such as Tom40 and Tom22 was not inhibited in por1D mitochondria, but even moderately enhanced (Figure S2E). TOM and TIM22 complexes and the steady-state levels of small Tim proteins were not disturbed in the por1D mitochondria used (Figures 2D and S2F), excluding that reduced levels of the translocases or small TIM chaperones led to an impaired carrier import in the absence of Por1. We conclude that por1D mitochondria are selectively impaired in the import of carrier precursors.

lite carriers. Porin forms an anion-preferring channel for the flux of a variety of metabolites and ions across the outer membrane. To address whether the characteristic anion selectivity was required for the carrier import, we selected POR1 point mutants that revealed a strongly reduced anion selectivity (Por1K19E) or even a cation selectivity (Por1K19,61E) (Blachly-Dyson et al., 1990). Metabolite transport across the outer membrane was determined in organello by adding NADH to isolated mitochondria and measuring its oxidation by the external NADH dehydrogenase of the inner membrane (Figure 3A) (Lee et al., 1998). NADH oxidation was virtually blocked in por1D mitochondria (Lee et al., 1998) and significantly reduced in Por1K19E and Por1K19,61E mitochondria (Figure 3A). Expression of these POR1 variants restored growth of por1D and por1D tim18D cells (Figures S3A and S3B), and the import of carrier precursors was not inhibited in Por1K19E and Por1K19,61E mitochondria but even slightly increased (Figures 3B and S3C), demonstrating that an impaired metabolite transport by porin did not disturb the biogenesis of carrier proteins. Second, we performed co-immunoprecipitation from lysed cells containing hemagglutinin (HA)-tagged porin. Porin co-purified Tom proteins, Om45, and mature carrier proteins as re€ ller ported (Figures 3C, S3D, and S3E) (Lauffer et al., 2012; Mu et al., 2016). Importantly, we observed a co-precipitation of the TIM22 complex subunits Tim18, Tim22, and Tim54 with porin, whereas the more abundant subunits of the TIM23 complex and Mia40 of the mitochondrial intermembrane space import and assembly system (Morgenstern et al., 2017) were not detected in the elution samples (Figure 3C). It has been reported that the human TIM22 complex interacts with the TOM complex with low efficiency (Kang et al., 2016). Similarly, we observed a low efficient co-purification of TIM22 subunits with HA-tagged Tom40 in yeast (Figure S3D). A direct comparison of co-precipitation of TIM22 subunits with Por1HA or Tom40HA revealed a considerably better TIM22-porin interaction than TIM22-TOM interaction (Figure S3D), in agreement with the observation that the human carrier translocase interacted more efficiently with VDAC than with TOM (Kang et al., 2016, Figure 1 source data therein). Upon cross-linking with the homo-bifunctional aminereactive reagent disuccinimidyl glutarate (DSG) to stabilize dynamic interactions, we observed an interaction of Por1 with the small Tim proteins Tim9 and Tim10 (Figure 3D). We conclude that porin/VDAC is an outer membrane interaction partner of the carrier translocase and the small TIM chaperones.

Distinct Functions of Porin in Transport of Metabolites and Carrier Proteins We asked how porin can affect the import of carrier precursors. First, we considered the possibility that the transport of metabolites via porin may indirectly influence the biogenesis of metabo-

Porin Promotes Carrier Transfer to the Inner Membrane To determine the protein import step that is promoted by porin, we dissected the carrier import pathway into distinct stages (Figure 1A) (Ryan et al., 1999; Rehling et al., 2003). Carrier precursors synthesized in the cytosol are bound to ATP-dependent

Figure 2. Porin Is Required for the Efficient Import of Carrier Proteins (A) 35S-labeled AAC and DIC were imported into wild-type (WT) and por1D mitochondria (from YPH499 yeast) and analyzed by blue native electrophoresis and autoradiography. Import reactions into mitochondria isolated from BY4741 and M3 yeast yielded comparable import defects in por1D mitochondria. (B) 35S-labeled AAC was imported in WT, por1D, and por1D + POR1 mitochondria and analyzed by blue native electrophoresis and autoradiography. (C) 35S-labeled Su9-DHFR, F1b, and cytochrome c1 (Cyt c1) were imported into WT and por1D mitochondria and analyzed by SDS-PAGE and autoradiography. (D) WT and por1D mitochondria were lysed with digitonin, and protein complexes were analyzed by blue native electrophoresis and immunodetection. See also Figure S2.

4 Molecular Cell 73, 1–10, March 7, 2019

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Figure 3. Distinct Functions of Porin in Protein and Metabolite Transport (A) Oxidation of externally added NADH by isolated yeast mitochondria. Upper panel, scheme of the assay. Middle panel, NADH oxidation in por1 mutant mitochondria was measured for the indicated periods. Lower panel, quantification of the NADH oxidation rate in the indicated mutant mitochondria. Depicted are mean values ± SEM of three independent experiments. (B) 35S-labeled AAC and DIC were imported into por1 mutant mitochondria and analyzed by blue native electrophoresis and autoradiography. (C) Wild-type (WT) and Por1HA total cell extracts were lysed with digitonin and subjected to affinity purification. Load (1%) and elution fraction (100%) were analyzed by SDS-PAGE and immunodetection. (D) Wild-type (WT) and Por1His mitochondria were treated with DSG followed by lysis with digitonin and affinity purification via Por1His. Load (5%) and elution (100%) fractions were analyzed by SDS-PAGE and immunodetection. Arrowhead, non-specific reaction (at the abundant Por1). See also Figure S3.

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Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

(legend on next page)

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Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

cytosolic chaperones (stage I). In the absence of ATP and Dc, the precursor-chaperone complexes preferentially accumulate at dimers of the receptor Tom70 on the mitochondrial surface (stage II), evidenced by a characteristic blue native band pattern (Figure 4A, lane 1) (Ryan et al., 1999; Wiedemann et al., 2001; Young et al., 2003). Upon addition of ATP, the precursor is released and translocated through the TOM channel to the small TIM chaperones in the intermembrane space (stage III), where the precursor is protected against proteases added to mitochondria (due to the absence of Dc, the precursor is not inserted into the inner membrane) (Figures 4B, lanes 1–3, and S4A, lane 5) (Ryan et al., 1999; Wiedemann et al., 2001; Truscott et al., 2002). In por1D mitochondria, accumulation of the [35S] AAC precursor at stage II was only mildly affected (Figures 4A). Formation of stage III and binding of the precursor to small TIM chaperones were moderately reduced in the absence of Por1 (Figures 4B and S4A). Thus, the lack of Por1 only moderately impairs the early carrier import stages of precursor recognition on the mitochondrial surface and translocation across the outer membrane. To study transfer of the carrier precursor from the intermembrane space into the inner membrane, we first accumulated carrier precursors at stage III by depletion of Dc. Subsequently, the membrane potential was restored and the precursors were chased to the TIM22 complex (stage IV) and inserted into the inner membrane (stage V) (Figure 4C) (Ryan et al., 1999; Rehling et al., 2003). The TIM22-bound stage IV intermediate can be directly visualized by blue native electrophoresis as shown by the mobility shift of the stage IV precursor band upon tagging of Tim18 (Figure 4C, lanes 13–15). por1D mitochondria were impaired in formation of the stage IV intermediate and the fully imported stage V carrier (Figures 4C, lanes 6–8 and 22–24, and S4B). Since the TIM22 complex is fully assembled in por1D mitochondria (Figure 2D), the lack of porin does not destabilize the translocase itself. We conclude that the lack of Por1 retards

the late stages of the carrier import that are required for carrier transport to the inner membrane. We asked whether and when porin interacts with carrier precursors during their import. Upon precursor accumulation at distinct import stages, the mitochondria were lysed and subjected to affinity purification via tagged Por1. Porin preferentially co-purified carrier precursor arrested in the intermembrane space (stage III: +ATP/–Dc) (Figure 4D). To exclude the possibility that the association of carrier precursor and porin occurred after lysis of the mitochondria, we performed chemical crosslinking in intact mitochondria with DSG. The best cross-linking efficiency between the 35S-labeled carrier precursor and porin was observed when the precursor was accumulated at stage III (Figure 4E). Upon arrest at stage III, AAC precursors formed specific cross-linking products with Tim10 (Figure S4C) that could be co-purified with Por1His under native conditions (Figure 4F). Taken together, porin interacts with carrier precursors accumulated at the small TIM chaperones in the intermembrane space and supports the transfer of the precursors to the inner membrane. DISCUSSION We report an unexpected function of the major mitochondrial metabolite channel. The b-barrel protein porin of the outer membrane plays a specific role in the import of a-helical metabolite carriers into the inner membrane (Figure 4G). The carrier precursors are recognized on the mitochondrial surface and have to pass the outer membrane; however, these steps are only mildly affected in porin-defective mitochondria. Instead, porin promotes the transfer of carrier precursors from the intermembrane space to the carrier translocase TIM22 of the inner membrane. How can an outer membrane protein perform this remarkable task? First, porin interacts with carrier precursors at the stage

Figure 4. Porin Binds Carrier Precursors and Promotes Transfer to the TIM22 Complex (A) Left panel: 35S-labeled AAC was arrested at stage II in wild-type (WT) and por1D mitochondria and chased to stage III where indicated. The import was analyzed by blue native electrophoresis and autoradiography. Right panel: quantification of stage II. WT value was set as control. Depicted are mean values ± SEM of three independent experiments. (B) Left panel: [35S]AAC was imported to stage III followed by treatment with proteinase K. Mitochondria were lysed with digitonin and analyzed by SDS-PAGE and autoradiography. Right panel: quantification of stage III formed in WT and por1D mitochondria. The import into WT mitochondria after 12 min was set as control. Depicted are mean values ± SEM of five independent experiments. (C) Lanes 1–8 and 17–24: 35S-labeled PiC and AAC were arrested in stage III upon depletion of Dc in WT and por1D mitochondria and chased to stages IV and V. The import was analyzed by blue native electrophoresis and autoradiography. Right panel: quantification of AAC at stage V. The WT value after 20 min was set as control. Depicted are mean values ± SEM of three independent experiments. Lanes 9–16: [35S]PiC was imported into WT and protein-A-tagged Tim18 (Tim18PA) mitochondria. Arrowhead, non-specific band. (D) Left panel: 35S-labeled AAC was accumulated at stages II, III, and V in WT and Por1HA mitochondria followed by affinity purification via Por1HA. Load (1%) and elution (100%) fractions were analyzed by SDS-PAGE and autoradiography. Right panel: quantification of AAC co-purified with Por1HA. The mean amount of co-purified AAC arrested at stage III (+ATP/Dc) was set as control. Depicted are mean values ± SEM of three independent experiments. The import arrest for stage II (–ATP/Dc) is not completely tight, and thus a fraction of precursors also accumulates at stage III under these conditions (Ryan et al., 1999), likely explaining the co-purification with Por1HA. (E) 35S-labeled AAC was accumulated at stages II, III, and V in WT and Por1His mitochondria followed by cross-linking with DSG and affinity purification via Por1His under denaturing conditions. Load (1%) and elution (100%) fractions were analyzed by SDS-PAGE and autoradiography. Por1xAAC, cross-linking product between porin and [35S]AAC. (F) 35S-labeled AAC was arrested at stage III in WT and Por1His mitochondria, followed by cross-linking with DSG and affinity purification via Por1His under native conditions. Load (1%) and elution (100%) fractions were analyzed by SDS-PAGE and autoradiography. (G) Hypothetical model of the role of porin in the carrier import pathway. Porin interacts with carrier precursors accumulated at small TIM chaperones in the intermembrane space (IMS) and recruits the TIM22 complex, thus facilitating precursor transfer to the inner membrane (IM). OM, outer membrane. See also Figure S4.

Molecular Cell 73, 1–10, March 7, 2019 7

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

III intermediate where the precursors are bound to small TIM chaperones in the intermembrane space. Second, porin binds the TIM22 complex and thus recruits the inner membrane translocase to the stage III intermediate, facilitating transfer of the precursor to TIM22 (Figure 4G). We conclude that porin functions as coupling factor in the carrier import pathway. The porin-TIM22 interaction represents a contact site between the mitochondrial outer and inner membranes. Porin/VDAC has been linked to several metabolic and signaling processes, including apoptosis, interaction with enzymes and cytoskeletal proteins, metabolic reprogramming during tumorigenesis, nucleic acid transport, lipid metabolism, and Ca2+ transfer via mitochondria-endoplasmic reticulum contact sites (Colombini, 2012; Lauffer et al., 2012; Messina et al., 2012; Campo et al., 2017; Miyata et al., 2018). The role of porin in promoting protein import to the inner membrane reveals a function in mitochondrial protein biogenesis. Metabolite translocation by porin is not required for its role in protein biogenesis, and porin does not generally stimulate protein import as the import of presequence-containing preproteins occurs independently of porin. By interacting with carrier precursors and the TIM22 complex, porin functions as specific coupling factor in the import pathway of inner membrane metabolite carriers. Porin thus performs mechanistically distinct functions as channel for numerous small molecules and ions and as outer-inner membrane coupling factor. The coupling function of porin in the carrier import reveals a principle for the regulation of the mitochondrial protein import apparatus. Whereas the copy numbers of respiratory complexes increase more than 3-fold when yeast cells are shifted from glucose to glycerol, the copy numbers of protein import components such as Tom70/Tom40, small TIMs, and Tim22/Tim54 are only marginally increased (mean factor of 1.15) (Morgenstern et al., 2017). Substantial changes of the levels of an import component have only been reported in a few cases such as the stress-regulated isoform Tim17A in metazoa (Rainbolt et al., 2013). How is then mitochondrial protein import regulated? In addition to differential expression and modification of individual precursor proteins in the cytosol, two main principles have been described that operate at the level of the import apparatus. First, the energetic activity of mitochondria can affect the Dcdependent protein import into the inner membrane and an ATP-dependent differential processing of proteins. Second, posttranslational modification of import components can regulate the efficiency of protein import, as shown with the phosphorylation of TOM subunits under different metabolic conditions and cell-cycle phases (Harbauer et al., 2014). Third, we propose that coupling factors that modulate the efficiency of an import pathway can serve as regulators when the levels of the coupling factor are adjusted under different growth conditions. The levels of porin are strongly dependent on the metabolic conditions; they increase nearly 3-fold when cells are shifted from glucose to glycerol (Figure S4D) (Morgenstern et al., 2017). Thus, regulating the levels of the outer membrane metabolite channel may serve as a means to regulate the efficiency of the import machinery for metabolite carriers of the inner membrane. In summary, porin of the mitochondrial outer membrane interacts with the carrier translocase of the inner membrane and

8 Molecular Cell 73, 1–10, March 7, 2019

facilitates the transfer of carrier precursors from the intermembrane space to the inner membrane. Porin thus plays a specific stimulatory role in the biogenesis of mitochondrial metabolite carriers. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Yeast strains B Plasmid construction B Preparation of yeast total cell extracts B Isolation of mitochondria B Protein import into isolated mitochondria B Experimental dissection of the carrier import pathway B Chemical cross-linking B Affinity purification B Polyacrylamide gel electrophoresis B Immunoblotting B Outer membrane permeability assay B Phospholipid analysis B Membrane potential measurements QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and one table and can be found with this article online at https://doi.org/10.1016/j.molcel.2018.12.014. ACKNOWLEDGMENTS We thank Dr. Michael Forte for the por1D yeast strain (M3 background) and Nicole Zufall for expert technical assistance. Work included in this study has also been performed in partial fulfillment of the requirements for the doctoral theses of L.E., K.N.D., and C.U.M. at the University of Freiburg. This work was supported by the Deutsche Forschungsgemeinschaft (BE 4679/2-1; PF 202/ 8-1 and 202/9-1), Sonderforschungsbereich 746, Research Training Group RTG 2202, and Excellence Initiative/Strategy of the German Federal & State Governments (EXC 294 BIOSS; GSC-4 Spemann Graduate School; EXC 2189 CIBSS Project ID 390939984). AUTHOR CONTRIBUTIONS L.E., M.P.D., K.N.D., C.U.M., and A.F. performed experiments and analyzed data together with T.B. and N.P.; T.B., N.P., and M.L.C. designed the concepts; T.B. and N.P. supervised the project; L.E., K.N.D., and T.B. prepared the figures; L.E., N.P., and T.B. wrote the manuscript; and all authors discussed results from the experiments and commented on the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: June 17, 2018 Revised: November 9, 2018 Accepted: December 14, 2018 Published: February 6, 2019

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SUPPORTING CITATIONS €brich The following reference appears in the Supplemental Information: Ku et al. (1998).

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€ger, V., Becker, T., Becker, L., Montilla-Martinez, M., Ellenrieder, L., Vo¨gtle, Kru F.N., Meyer, H.E., Ryan, M.T., Wiedemann, N., Warscheid, B., et al. (2017). Identification of new channels by systematic analysis of the mitochondrial outer membrane. J. Cell Biol. 216, 3485–3495.

Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G.Y., Labouesse, M., MinvielleSebastia, L., and Lacroute, F. (1991). A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7, 609–615.

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Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H., and Hieter, P. (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122. Colombini, M. (2012). VDAC structure, selectivity, and dynamics. Biochim. Biophys. Acta 1818, 1457–1465. de Marcos-Lousa, C., Sideris, D.P., and Tokatlidis, K. (2006). Translocation of mitochondrial inner-membrane proteins: Conformation matters. Trends Biochem. Sci. 31, 259–267. ski, q., Becker, L., Kru €ger, V., Mirus, O., Straub, S.P., Ellenrieder, L., Opalin Ebell, K., Flinner, N., Stiller, S.B., Guiard, B., et al. (2016). Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10. Nat. Commun. 7, 13021. Endo, T., and Yamano, K. (2009). Multiple pathways for mitochondrial protein traffic. Biol. Chem. 390, 723–730. Endres, M., Neupert, W., and Brunner, M. (1999). Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex. EMBO J. 18, 3214–3221. Gebert, N., Gebert, M., Oeljeklaus, S., von der Malsburg, K., Stroud, D.A., Kulawiak, B., Wirth, C., Zahedi, R.P., Dolezal, P., Wiese, S., et al. (2011). Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane. Mol. Cell 44, 811–818. Gietz, R.D., and Woods, R.A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96. Haan, C., and Behrmann, I. (2007). A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background. J. Immunol. Methods 318, 11–19. Harbauer, A.B., Zahedi, R.P., Sickmann, A., Pfanner, N., and Meisinger, C. (2014). The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease. Cell Metab. 19, 357–372. Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop, M. (2004). A versatile toolbox for PCR-based tagging of yeast genes: New fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962. Jiang, F., Ryan, M.T., Schlame, M., Zhao, M., Gu, Z., Klingenberg, M., Pfanner, N., and Greenberg, M.L. (2000). Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function. J. Biol. Chem. 275, 22387–22394. Kang, Y., Baker, M.J., Liem, M., Louber, J., McKenzie, M., Atukorala, I., Ang, C.-S., Keerthikumar, S., Mathivanan, S., and Stojanovski, D. (2016). Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability. eLife 5, e17463.

€bert, K., Czupalla, C., Pursche, T., Hoflack, B., Ro¨del, G., and Lauffer, S., Ma Krause-Buchholz, U. (2012). Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p. J. Biol. Chem. 287, 17447–17458. Lee, A.C., Xu, X., Blachly-Dyson, E., Forte, M., and Colombini, M. (1998). The role of yeast VDAC genes on the permeability of the mitochondrial outer membrane. J. Membr. Biol. 161, 173–181. Lionaki, E., de Marcos Lousa, C., Baud, C., Vougioukalaki, M., Panayotou, G., and Tokatlidis, K. (2008). The essential function of Tim12 in vivo is ensured by the assembly interactions of its C-terminal domain. J. Biol. Chem. 283, 15747–15753. Longtine, M.S., McKenzie, A., 3rd, 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. Messina, A., Reina, S., Guarino, F., and De Pinto, V. (2012). VDAC isoforms in mammals. Biochim. Biophys. Acta 1818, 1466–1476. Miyata, N., Fujii, S., and Kuge, O. (2018). Porin proteins have critical functions in mitochondrial phospholipid metabolism in yeast. J. Biol. Chem. 293, 17593–17605. €bbert, P., Peikert, C.D., Dannenmaier, S., Morgenstern, M., Stiller, S.B., Lu Drepper, F., Weill, U., Ho¨ß, P., Feuerstein, R., Gebert, M., et al. (2017). Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Rep. 19, 2836–2852. €ller, C.S., Bildl, W., Haupt, A., Ellenrieder, L., Becker, T., Hunte, C., Fakler, Mu B., and Schulte, U. (2016). Cryo-slicing blue native-mass spectrometry (csBNMS), a novel technology for high-resolution complexome profiling. Mol. Cell. Proteomics 15, 669–681. Neupert, W., and Herrmann, J.M. (2007). Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749. Palmieri, F., and Pierri, C.L. (2010). Mitochondrial metabolite transport. Essays Biochem. 47, 37–52. Peixoto, P.M.V., Gran˜a, F., Roy, T.J., Dunn, C.D., Flores, M., Jensen, R.E., and Campo, M.L. (2007). Awaking TIM22, a dynamic ligand-gated channel for protein insertion in the mitochondrial inner membrane. J. Biol. Chem. 282, 18694–18701. Qiu, J., Wenz, L.-S., Zerbes, R.M., Oeljeklaus, S., Bohnert, M., Stroud, D.A., Wirth, C., Ellenrieder, L., Thornton, N., Kutik, S., et al. (2013). Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation. Cell 154, 596–608. Rainbolt, T.K., Atanassova, N., Genereux, J.C., and Wiseman, R.L. (2013). Stress-regulated translational attenuation adapts mitochondrial protein import through Tim17A degradation. Cell Metab. 18, 908–919.

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10 Molecular Cell 73, 1–10, March 7, 2019

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Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-AAC (all isoforms)

Schuler et al., 2016

GR3614-3

Rabbit polyclonal anti-Atp4

Ellenrieder et al., 2016

GR1970-5

Rabbit polyclonal anti-Cox13

Morgenstern et al., 2017

GR1543-3

Rabbit polyclonal anti-DIC

Stiller et al., 2016

GR2055-KB

Rabbit polyclonal anti-Mia40

Stiller et al., 2016

B315-7

Rabbit polyclonal anti-Mim1

Ellenrieder et al., 2016

GR544-1

Rabbit polyclonal anti-Mir1 (PiC)

Stiller et al., 2016

GR171-5

Rabbit polyclonal anti-mtHsp60

Schuler et al., 2016

170 (7/9/98)

Rabbit polyclonal anti-mtHsp70

Schuler et al., 2016

GR1830-3

Rabbit polyclonal anti-Oac1

This paper

GR3075-4

Rabbit polyclonal anti-Mge1

Schuler et al., 2016

GR1837-5

Rabbit polyclonal anti-Om45

Ellenrieder et al., 2016

GR1311-4

Rabbit polyclonal anti-Por1

Ellenrieder et al., 2016

94D

Rabbit polyclonal anti-Tim9

Stiller et al., 2016

GR2013-7

Rabbit polyclonal anti-Tim10

Ellenrieder et al., 2016

GR2040-4

Rabbit pre-immune serum (Tim10)

This paper

GR2040-PI

Rabbit polyclonal anti-Tim12

Stiller et al., 2016

GR906-7

Rabbit polyclonal anti-Tim17

Schuler et al., 2016

GR1854-3

Rabbit polyclonal anti-Tim18

This paper

GR5114-3

Rabbit polyclonal anti-Tim22

This paper

GR5113-4

Rabbit polyclonal anti-Tim23

Stiller et al., 2016

GR133-8

Rabbit polyclonal anti-Tim44

Schuler et al., 2016

GR1836-4

Rabbit polyclonal anti-Tim50

Stiller et al., 2016

257-7

Rabbit polyclonal anti-Tim54

Schuler et al., 2016

GR2012-4

Rabbit polyclonal anti-Tom20

Ellenrieder et al., 2016

GR3225-7

Rabbit polyclonal anti-Tom22

Ellenrieder et al., 2016

GR3227-3

Rabbit polyclonal anti-Tom40

Ellenrieder et al., 2016

168-5

Rabbit polyclonal anti-Tom70

Ellenrieder et al., 2016

GR657-3

Goat Peroxidase-coupled anti-Rabbit IgG

Jackson ImmunoResearch Laboratories

RRID: AB_2313567; Cat. #111035-003

L-[35S]-Methionine

PerkinElmer

Cat. #NEG009005MC

[33P]Phosphoric acid

Hartmann Analytic

Code: FF-1

Disuccinimidyl glutarate (DSG)

Thermo Fisher Scientific

Cat. #20593

Dimethyl pimelidate dihydrochloride

Sigma

Cat. #D8388

Anti-HA affinity matrix

Roche

Cat. #11815016001

Ni-NTA Agarose

QIAGEN

Cat. #30230

Protein A Sepharose CL-4B

GE Healthcare

Cat. #17-0963-03

Antibodies

Chemicals, Peptides, and Recombinant Proteins

Critical Commercial Assays TNT Quick Coupled Reaction Mix

Promega

Cat. #L2080

Roti-Quant Bradford reagent

Roth

Cat. #K015.3

KOD Hot Start Master Mix

Merck Millipore

Cat. #71842-3

RedTaq Polymerase PCR Master Mix (2x)

Genaxxon Bioscience

Cat. #M3029.0500 (Continued on next page)

Molecular Cell 73, 1–10.e1–e7, March 7, 2019 e1

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

mMessage mMachine SP6 Transcription Kit

Thermo Fisher Scientiic

Cat. #AM1340

MEGAclear Transcription Clean-Up Kit

Thermo Fisher Scientiic

Cat. #AM1908

YPH499 (WT) MATa ura3-52 lys2-801_amber ade2-101_orchre trp1-D63 his3-D200 leu2-D1

Sikorski and Hieter, 1989

1501

YPH499 (WT) pFL39 (empty vector)

This paper

5260

YPH499 (WT) pRS424 (empty vector)

This paper

4449

YPH499 por1::HIS3MX6

This paper

4891

Experimental Models: Organisms/Strains

YPH499 por1::HIS3MX6 pFL39 (empty vector)

This paper

5261

YPH499 tim18::kanMX4

Wagner et al., 2008

1383

YPH499 tom70::HIS3MX6

Qiu et al., 2013

1059

YPH499 sdh3::HIS3MX6

Gebert et al., 2011

2709

YPH499 por1::hphNT1 tim18::kanMX4

This paper

5265

YPH499 por1::hphNT1 tom70::kanMX4

This paper

5267

YPH499 por1::hphNT1 sdh3::HIS3MX6

This paper

5332

YPH499 tim18::TIM18ProtA-HIS3MX6

Rehling et al., 2003

1375

YPH499 por1::POR1HA-HIS3MX6

€ller et al., 2016 Mu

3716

YPH499 por1::HIS3MX6 pFL39-POR1

This paper

5262

YPH499 por1::HIS3MX6 pFL39-por1K19E

This paper

5263

YPH499 por1::HIS3MX6 pFL39-por1K19,61E

This paper

5264

YPH499 por1::hphNT1 tim18::kanMX4 pFL39 (empty vector)

This paper

5268

YPH499 por1::hphNT1 tim18::kanMX4 pFL39-POR1

This paper

5269

YPH499 por1::hphNT1 tim18::kanMX4 pFL39-por1K19E

This paper

5270

YPH499 por1::hphNT1 tim18::kanMX4 pFL39-por1K19,61E

This paper

5271

YPH499 tim10::tim10-2

Truscott et al., 2002

1390

YPH499 pRS424-POR1

This paper

5272

BY4741 (WT) MATa his3D1 leu2D0 met15D0 ura3D0

EUROSCARF

1354

BY4741 por1::POR1His-HIS3MX6

This paper

5273

BY4741 por1::KanMX4

EUROSCARF

4865

BY4741 tom40::TOM40HA-HIS3MX6

This paper

5035

M3 (WT) MATa lys2 his4 trp1 ade2 leu2 ura3

Blachly-Dyson et al., 1997

1280

M3 por1::LEU2

Blachly-Dyson et al., 1997

1281

See Table S1

See Table S1

pGEM4Z-AAC (Neurospora crassa)

Pfanner/Becker Labs

A01

pGEM4Z-DIC (DIC1) (S. cerevisiae)

Pfanner/Becker Labs

A32

pGEM4Z-PiC (MIR1) (S. cerevisiae)

Pfanner/Becker Labs

A24

pGEM4Z-Su9-DHFR (Su9 (1-69, N. crassa)-DHFR (mouse))

Pfanner/Becker Labs

S02

pGEM4Z-F1b (S. cerevisiae)

Pfanner/Becker Labs

F01

pGEM4Z-Cyt. c1 (N. crassa)

Pfanner/Becker Labs

C06

pGEM4Z-Tom40 (S. cerevisiae)

Pfanner/Becker Labs

1495

pFL39

Bonneaud et al., 1991

X15

pFL39-POR1 (S. cerevisiae)

Qiu et al., 2013

1696

pFL39-por1K19E

This paper

3088

pFL39-por1K19,61E

This paper

3091

pRS424

Christianson et al., 1992

X29

pRS424-POR1 (S. cerevisiae)

This paper

3084

Oligonucleotides See Table S1 Recombinant DNA

(Continued on next page)

e2 Molecular Cell 73, 1–10.e1–e7, March 7, 2019

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

pFA6a-3xHA-HIS3MX6

Knop et al., 1999

1450; pYM2

pFA6a-His10-HIS3MX6

Qiu et al., 2013

X63

pFA6a-HIS3MX6

Longtine et al., 1998

1424

pFA6a-hphNT1

Janke et al., 2004

2722

ImageJ

National Institutes of Health, USA

https://imagej.nih.gov/ij/

Multi Gauge v.3.2

FujiFilm

N/A

Software and Algorithms

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nikolaus Pfanner ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Yeast strains used in this study are derivatives of the S. cerevisiae strains YPH499, BY4741 and M3, which are listed with their genotypes in KEY RESOURCES TABLE. Yeast cells were grown in YP medium (1% [w/v] yeast extract; 2% [w/v] bacto-peptone) or selective complete medium (SM) (0.67% [w/v] yeast nitrogen base; 0.07% [w/v] amino acid mixture) supplemented with 2% [w/v] glucose (YPD, SMD), 3% [w/v] glycerol (YPG) or 3% [w/v] glycerol and 0.1% [w/v] glucose (SMG). Cells were grown at 23-37
C to an early exponential growth phase. The optical density of the culture was determined at a wavelength of 600 nm (OD600). METHOD DETAILS Yeast strains The yeast wild-type (WT) strains YPH499, BY4741 and M3 as well as the yeast mutant strains tim18D, tom70D, por1D (M3 background), sdh3D, tim10-2, Tim18ProtA and Por1HA have been described before (Blachly-Dyson et al., 1997; Truscott et al., 2002; Re€ller et al., 2016). The open reading frame of POR1 was hling et al., 2003; Wagner et al., 2008; Gebert et al., 2011; Qiu et al., 2013; Mu deleted in the YPH499 background by homologous recombination using DNA cassettes encoding a HIS3MX6 or hphNT1 selection marker (Longtine et al., 1998; Janke et al., 2004). The cassettes were amplified via PCR using KOD hot-start DNA polymerase (Merck Millipore). The utilized primers (see Table S1) added a sequence to the 50 end of the cassette homologous to 47 base pairs upstream of the start codon of POR1 and a sequence to the 30 end of the cassette that matches 47 base pairs downstream of the stop codon of POR1. In order to add a deca-histidine (His) tag to the C terminus of Por1, a DNA cassette encoding the tag as well as a HIS3MX6 selection marker (Qiu et al., 2013) was amplified via PCR (see Table S1 for primers) and introduced right in front of the stop codon of POR1 via homologous recombination. For C-terminal tagging of Tom40 with a hemagglutinin (HA) tag, we amplified a cassette encoding the tag plus a HIS3MX6 selection marker from the plasmid pYM2 (Knop et al., 1999) via PCR using the primer pair Tom40-HAfw/Tom40-HA-rv (Table S1). The cassette was introduced right in front of the stop codon of TOM40 via homologous recombination. DNA cassettes, plasmids encoding POR1 variants and the respective empty vector controls were transformed into parental strains following the lithium acetate method using carrier DNA (Gietz and Woods, 2002). Plasmid construction Plasmids and primers for plasmid construction used in this study are listed in KEY RESOURCES TABLE and Table S1, respectively. The centromeric plasmid pFL39 encoding wild-type POR1 under control of its endogenous promoter and terminator (Qiu et al., 2013) was used as template to generate pFL39-por1K19E and pFL39-por1K19,61E via site-directed mutagenesis, using the primer pairs Por1K19E-fw/Por1-K19E-rv and Por1-K61E-fw/Por1-K61E-rv, respectively (Table S1). For overexpression of Por1 the open reading frame (ORF) of POR1 together with its promoter and terminator was introduced into the multi-copy plasmid pRS424. The ORF of POR1 together with its endogenous promoter and terminator was amplified from S. cerevisiae genomic DNA (YPH499) by PCR using KOD hot-start DNA polymerase and the primer pair BamHI-Por1-up/SalI-Por1-down (Table S1). The primers added a BamHI restriction site to the 50 end and a SalI restriction site to the 30 end of the POR1 gene. The resultant PCR product was digested with BamHI and SalI (NEB) and ligated into BamHI/SalI digested pRS424 with the T4 DNA ligase (NEB), following the manufacturers’ recommendations. Preparation of yeast total cell extracts Proteins were extracted from yeast cells by the method of Kushnirov (2000). 2.5 to 10 OD600 cells were harvested by centrifugation (1,000 x g; 3 min; 20
C), washed with water and resuspended in 0.1 M NaOH (80 ml/OD600). After 5 min incubation at room

Molecular Cell 73, 1–10.e1–e7, March 7, 2019 e3

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

temperature cells were pelleted (15,000 x g; 1 min; 20
C), 1 OD600 cells were lysed with 60 ml SDS sample buffer (2% [w/v] SDS; 10% [v/v] glycerol; 0.01% [w/v] bromphenol blue; 0.2% [v/v] b-mercaptoethanol; 60 mM Tris/HCl, pH 6.8) and boiled for 5 min at 95
C. Insoluble material was removed by centrifugation (15,000 x g; 1 min; 20
C) and the supernatant was subjected to SDS-PAGE analysis. To obtain total cell extracts for affinity purification, cells were grown to early logarithmic phase in YPG at 30
C, harvested and washed with dH2O and lysis buffer (0.1 mM EDTA; 50 mM NaCl; 10% [v/v] glycerol; 20 mM Tris-HCl, pH 7.4). Excessive buffer was removed by centrifugation (1,500 x g; 10 min; 20
C) and the cell pellet was snap-frozen in liquid nitrogen before grinding using a cryo-mill at 25 Hz for 10 min (Ellenrieder et al., 2016). The resulting cell powder was stored at 80
C until further use. Isolation of mitochondria Mitochondria were isolated from yeast cells via differential centrifugation (Ellenrieder et al., 2016). Yeast cells were grown in YPG or selective medium with glycerol as carbon source at 30
C to early logarithmic growth phase to minimize alterations of the mitochondrial phospholipid composition, which were reported for por1D cells grown to saturation on fermentable carbon sources (upon depletion of both Por1 and Por2 and cell growth on fermentable medium to saturation, the levels of cardiolipin were strongly decreased; Miyata et al., 2018). Cells were harvested by centrifugation (5,300 x g; 8 min), washed with dH2O and the wet weight of the cell pellet was determined. The cells were then treated with 10 mM DTT in 100 mM Tris/H2SO4, pH 9.4 (2 mL/g cells) for 20 min at 30
C under constant shaking with 130 rpm. Cells were pelleted by centrifugation (2,000 x g; 5 min), washed with zymolyase buffer (1.2 M sorbitol; 20 mM KPi, pH 7.4) and incubated for 45 min at 30
C in zymolyase buffer (7 mL/g cells) containing 3 mg zymolyase per g cell. The generated spheroplasts were then pelleted by centrifugation (2,000 x g; 5 min; 20
C), washed with zymolyase buffer and resuspended in ice-cold homogenization buffer (0.6 M sorbitol; 1 mM EDTA; 0.2% [w/v] bovine serum albumin; 1 mM phenylmethylsulfonyl fluoride (PMSF); 10 mM Tris/HCl, pH 7.4) (6.5 mL/g cells). Cell homogenates were obtained by breaking up the spheroplasts mechanically using a glass-Teflon homogenizer. Cell debris and nuclei within the homogenate were removed by centrifugation (2,000 x g; 5 min; 4
C) followed by the isolation of mitochondria from the supernatant by centrifugation (13,000 x g; 10 min; 4
C). Mitochondria were resuspended in SEM buffer (250 mM sucrose; 1 mM EDTA; 10 mM MOPS-KOH, pH 7.2) and the last two centrifugation steps were repeated. Mitochondria were resuspended in a small volume of SEM buffer and protein concentration was determined photometrically by using the Roti-Quant Bradford reagent (Roth). The mitochondrial protein concentration was adjusted to 10 mg/mL with SEM buffer, aliquots were frozen in liquid nitrogen and stored at 80
C until further use. Protein import into isolated mitochondria Radiolabeled mitochondrial precursor proteins were synthesized in a coupled transcription/translation reaction using a cell-free system based on rabbit reticulocyte lysate. pGEM4Z plasmids encoding the respective open reading frames were used for the in vitro synthesis and are listed in KEY RESOURCES TABLE. In case of in vitro synthesized radiolabeled Tom22, the open reading frame (ORF) was amplified from yeast genomic DNA (YPH499) by PCR using RedTaq polymerase (Genaxxon bioscience) and the primer pair Tom22-SP6-fwd / Tom22-Sp6-rev (Table S1). The primer Tom22-Sp6-fwd added an SP6 promoter and Kozak sequence to the 50 end of the ORF. Subsequently, we synthesized the RNA utilizing the mMessage mMachine SP6 transcription kit (Thermo Fisher). The transcripts were purified using the MEGAclear transcription clean-up kit (Thermo Fisher) following the manufacturer’s recommendations. 2.5 mg pGEM4Z plasmid or 2 mg RNA encoding the desired ORF were mixed with 100 ml TNT Quick Coupled Reaction Mix (Promega) in the presence of 100 mCi [35S]methionine (PerkinElmer) and incubated for 90-120 min at 30
C. The reaction was stopped by adding 20 mM methionine as well as 250 mM sucrose (final concentrations). Radiolabeled proteins were used freshly or stored at 80
C until use. In a basic import reaction mitochondria corresponding to 50 mg protein content were resuspended in 100 ml import buffer (3% [w/v] bovine serum albumin; 250 mM sucrose; 80 mM KCl; 5 mM MgCl2; 5 mM methionine; 2 mM KH2PO4; 10 mM MOPS/KOH, pH 7.2) supplemented with 4 mM ATP, 4 mM NADH and 4%–8% [v/v] reticulocyte lysate containing the radiolabeled precursor protein. Where indicated, 10 mM reduced glutathione was added to the import reaction. In control reactions, NADH was omitted and a mixture of 8 mM antimycin A, 1 mM valinomycin and 20 mM oligomycin (AVO; final concentrations) was added to dissipate the membrane potential across the mitochondrial inner membrane. Samples were incubated at 25
C starting with the longest time point. Transfer on ice and addition of AVO to all samples stopped the import reaction. Where indicated, samples were treated with 50 mg/mL proteinase K for 15 min on ice to remove non-imported precursor proteins. The protease was inactivated by 10 min incubation with 1 mM PMSF on ice. Mitochondria were re-isolated by centrifugation (20,000 x g; 10 min; 4
C) and washed with SEM buffer. Where indicated, mitochondria were incubated in EM swelling buffer (1 mM EDTA; 10 mM MOPS-KOH, pH 7.2) to rupture the mitochondrial outer membrane. Mitochondrial proteins were analyzed by SDS-PAGE or blue native electrophoresis as outlined below. For affinity purification, the import reaction was scaled up 10- to 14-fold. After separation via SDS or blue native gel electrophoresis, imported proteins were detected on dried gels by digital autoradiography using the Typhoon FLA-9000 scanner (GE Healthcare). Images were analyzed using ImageJ software (NIH). Experimental dissection of the carrier import pathway In order to arrest carrier precursor proteins at distinct import stages, the energy content of the import reactions was modified according to published procedures (Ryan et al., 1999; Rehling et al., 2003). To deplete ATP from the import reaction, mitochondria

e4 Molecular Cell 73, 1–10.e1–e7, March 7, 2019

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

and reticulocyte lysate containing the radiolabeled precursor protein were pre-treated with 25 U/mL apyrase (Sigma Aldrich) for 10 min at room temperature. Carrier precursors were arrested in stage III by depletion of the membrane potential. To analyze the formation of stage III, mitochondria were treated with proteinase K as described above. Subsequently, mitochondrial membranes were solubilized in lysis buffer (0.1 mM EDTA; 50 mM NaCl; 10% [v/v] glycerol; 20 mM Tris-HCl, pH 7.4) containing 1% [w/v] digitonin for 15 min on ice. Insoluble material was removed by centrifugation and the supernatants were analyzed by SDS-PAGE. In order to reversibly dissipate the membrane potential, import samples that lacked NADH were supplemented with 80 mM carbonyl cyanide m-chlorophenyl hydrazone and 20 mM oligomycin. To restore the membrane potential in the chase reaction, mitochondria were resuspended in import buffer containing 4 mM ATP, 4 mM NADH, 10 mM creatine phosphate and 0.2 mg/mL creatine kinase. Chemical cross-linking For chemical cross-linking, isolated mitochondria were diluted in SEM buffer to a protein concentration of 1 mg/mL. The cross-linking reagent DSG (Pierce) was dissolved in dimethyl sulfoxide (DMSO) and added to a final concentration of 1 mM. After incubating the samples for 30 min on ice, cross-linking was quenched with 0.1 M Tris/HCl, pH 7.4. Mitochondria were re-isolated by centrifugation (20,000 x g; 10 min; 4
C), washed with SEM buffer and subjected to affinity purification as described below. Affinity purification To analyze interaction partners of Por1 and Tom40, we used cell extracts from yeast strains expressing HA-tagged variants of either Por1 or Tom40. We generated total cell extracts by cryo-grinding as described above. Lysis buffer containing 1% [w/v] digitonin was used to solubilize the cell powder (1 mL/100 mg powder) at 4
C for 45 min. After removal of insoluble material via centrifugation (20,000 x g; 10 min; 4
C) lysates were mixed with anti-HA affinity matrix (Roche) (1/20 bed volume) that was activated with 0.5 M acetate buffer pH 3.5 and equilibrated with lysis buffer containing 0.1% [w/v] digitonin. Samples were incubated for 90 min at 4
C. Subsequently, the affinity matrix was excessively washed with lysis buffer containing 1% [w/v] digitonin. We used SDS sample buffer to elute bound proteins under constant rotation for 5 min at room temperature. Load and elution samples were analyzed by SDS-PAGE and immunoblotting (see below). To analyze binding of the carrier precursors to porin at different import stages, the AAC precursor was arrested at different import stages in Por1HA mitochondria by manipulating the energy content in the import reaction as described above. Subsequently, mitochondria were lysed in lysis buffer containing 1% [w/v] digitonin (1 mg/mL protein concentration) at 4
C for 15 min. After removal of the insoluble material by centrifugation (20,000 x g; 10 min; 4
C), lysates were subjected to anti-HA affinity purification as described above. For affinity purification of cross-linking products via Por1His under denaturing conditions, mitochondria were lysed in lysis buffer containing 1% [w/v] SDS and 10 mM imidazole (1 mg/mL protein concentration) for 5 min at 95
C. Insoluble material was removed by centrifugation (20,000 x g; 10 min). The mitochondrial lysates were then diluted 1:10 in lysis buffer containing 0.2% [v/v] Triton X-100 and 10 mM imidazole and incubated with Ni2+-NTA agarose beads (QIAGEN) (1/100 bed volume) for 90 min at 4
C under constant rotation. After excessive washing of the affinity matrix with lysis buffer containing 0.2% [v/v] Triton X-100 and 40 mM imidazole, bound proteins were eluted with lysis buffer containing 0.2% [v/v] Triton X-100 and 250 mM imidazole. For co-immunoprecipitation, antibodies (KEY RESOURCES TABLE) were cross-linked with 7 mM dimethyl pimelidate (Sigma) to protein A Sepharose (GE Healthcare) in the presence of 0.1 M sodium tetraborate for 30 min at 20
C. Subsequently, cross-linking was quenched with 0.2 M Tris/HCl, pH 8.0 for 2 h at 4
C. The affinity matrices were stored in phosphate buffered saline at 4
C until further use. Co-immunoprecipitation experiments were performed following the protocol of affinity purifications via HA-tagged proteins. Bound proteins were eluted from the co-immunoprecipitation column with 0.1 M glycine/HCl, pH 2.5, followed by immediate neutralization with 1 M Tris-base. Polyacrylamide gel electrophoresis Protein samples were denatured in SDS sample buffer for 5 min at 95
C and analyzed by SDS-PAGE. Samples were loaded onto selfcast gels (10% [w/v] acrylamide; 0.31% [w/v] bis-acrylamide; 333 mM Bis-Tris, pH 6.4) and electrophoresis was performed in the presence of MES gel running buffer (100 mM 2-N-morpholino ethane sulfonic acid (MES); 100 mM Tris; 0.1% [w/v] SDS; 2.05 mM EDTA, pH 7.3) at 90 mA for approximately 2 h. Blue native electrophoresis was used to analyze mitochondrial protein complexes. Mitochondrial membranes were solubilized with lysis buffer containing 1% [w/v] digitonin (1 mg/mL protein concentration) for 15 min at 4
C. After removal of insoluble material (16,100 x g; 10 min; 4
C), BN sample buffer (0.5% [w/v] Coomassie G; 50 mM ε-amino n-caproic acid; 10 mM Bis-Tris/HCl, pH 7.0) was added to the supernatant. The samples were loaded onto self-cast blue-native gradient gels (6%–13% [w/v] acrylamide; 0.19%–0.40% [w/v] bis-acrylamide; 67 mM ε-amino n-caproic acid; 50 mM Bis-Tris/HCl, pH 7.0). Electrophoresis was performed for 90-110 min at 15 mA in the presence of BN cathode buffer (0.02% [w/v] Coomassie G; 50 mM Tricine; 15 mM Bis-Tris/HCl, pH 7.0) and anode buffer (50 mM Bis-Tris/HCl, pH 7.0). For western blotting analyses, the BN cathode buffer was replaced by a cathode buffer lacking Coomassie G after 30 min of electrophoresis.

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Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

Immunoblotting Proteins were blotted from polyacrylamide gels to polyvinylidene fluoride (PVDF) membranes (EMD Millipore) by semi-dry transfer in blotting buffer (20% [v/v] MeOH; 150 mM glycine; 0.02% [w/v] SDS; 20 mM Tris base) at 250 mA for 2 h. Protein-free areas were blocked with 5% [w/v] skimmed milk powder in TBS buffer (12.5 mM NaCl; 20 mM Tris/HCl, pH 7.4) for 1 h. Membranes were then incubated with the first antibody (see KEY RESOURCES TABLE) at room temperature for 2 h or at 4
C over night. After excessive washing with TBS, membranes were incubated with horseradish peroxidase-coupled anti-rabbit secondary antibody (Jackson) at room temperature for 1 h. Antibody signals were detected using enhanced chemiluminescence (Haan and Behrmann, 2007) and the image reader LAS3000 (FujiFilm). The signal specificities of the antibodies were confirmed by their loss when mitochondria from corresponding deletion strains were loaded. In case of essential genes, mitochondria from strains expressing respective variants were loaded and a size-shift of the immunosignal revealed specificity of the used antisera. Images were analyzed using Multi Gauge (FujiFilm). Outer membrane permeability assay We used oxidation of exogenously added NADH by isolated mitochondria as a parameter to determine outer membrane permeability (Lee et al., 1998). Isolated mitochondria were diluted in SEM buffer to a final protein concentration of 40 mg/mL. NADH was added to a final concentration of 30 mM to start the oxidation reaction. The absorbance of NADH at 340 nm was determined photometrically with a Lamda 35 UV/VIS spectrometer (PerkinElmer) in intervals of 5 s over a period of 10 min. Due to oxidation of NADH the absorbance at 340 nm decreased over time. We calculated the concentration of NADH based on the absorbance and its molar extinction coefficient (6.22 3 103 l x mol1 x cm1). Within the linear range of NADH oxidation (0 to 120 s), the oxidation rate was determined on basis of the slope and normalized to the amount of mitochondria (protein content) applied. Phospholipid analysis We analyzed mitochondrial phospholipids essentially as described before (Ellenrieder et al., 2016). Wild-type and por1D yeast strains were grown in YPG to an early logarithmic growth phase at 30
C. 25 OD600 cells were harvested by centrifugation and resuspended in YPG to a final concentration of 5 OD600/mL. The cell suspension was then incubated in the presence of 70 mCi [33P]phosphate (Hartmann Analytics) for 90 min at 30
C under constant shaking at 130 rpm. Cells were harvested by centrifugation (1,000 x g; 3 min; 20
C), washed with dH2O and mitochondria were isolated by differential centrifugation. Lipids from mitochondria corresponding to 250 mg – 500 mg protein content were extracted with 4 mL 2:1 chloroform/methanol [v/v] for 1 h under vigorous shaking conditions. The extract was washed successively with 2 mL 0.034% (w/v) MgCl2, 2 mL 4:1 2N KCl/methanol (v/v) and 1.5 mL 48:47:3 methanol/H2O/chloroform [v/v/v] before lipids were dried under constant nitrogen flow. Lipids were solved in 50 ml 2:1 chloroform/methanol (v/v) and applied to thin-layer chromatography (TLC) Silica gel 60 plates (Merck). Before use, the TLC plate was shortly soaked in 1.8% [w/v] boric acid solved in ethanol and baked for 15 min at 100
C. Lipids were separated for 150 min using 30:35:7:35 chloroform/ ethanol/water/triethylamine [v/v/v/v] as solvent. Subsequently, the plate was dried and labeled phospholipids were detected by digital autoradiography using the Typhoon FLA-9000 scanner (GE Healthcare). Images were analyzed using ImageJ software (NIH). Co-migration of phospholipid standards (Avanti) was used to identify specific phospholipid bands. Membrane potential measurements The membrane potential of the mitochondrial inner membrane was assessed by quenching of the fluorescent dye 3,3-dipropylthiadicarbocyanine iodide (DiSC3) upon voltage-dependent uptake into mitochondria (Schuler et al., 2016). In brief, the fluorescence of 2 mM DiSC3 in potential buffer (0.1% [w/v] bovine serum albumin; 0.6 M sorbitol; 10 mM MgCl2; 0.5 mM EDTA; 20 mM KPi, pH 7.4) was determined using a 650-40 fluorescence spectrophotometer (Perkin Elmer) at 25
C with excitation at 622 nm, emission at 670 nm and slits at 5 nm. Fluorescence was set to 8 arbitrary units and the measurement was started. After 20 s mitochondria were added to a final protein concentration of 20 mg/mL and fluorescence quenching was recorded. After 180 s the membrane potential was dissipated using 1 mM valinomycin and measurement was continued to a total of 300 s. QUANTIFICATION AND STATISTICAL ANALYSIS In Figure 3A, mean values of the NADH oxidation rate (nmol x min1 x mg1) of wild-type and POR1 mutant mitochondria from three independent experiments were determined. Error bars represent the standard error of the mean. In Figure 4A, the amounts of [35S]AAC arrested in stage II in wild-type and por1D mitochondria were quantified from three independent import experiments using Multi Gauge software (FujiFilm). The formation of stage II in wild-type mitochondria was set to 100%. Error bars represent the standard error of the mean. In Figure 4B, the amounts of [35S]AAC arrested in stage III in wild-type and por1D mitochondria after 3, 6 and 12 min were quantified from five independent import experiments using Multi Gauge software (FujiFilm). The formation of stage III in wild-type mitochondria after 12 min import was set to 100%. Error bars represent the standard error of the mean. In Figure 4C, the amounts of [35S]AAC assembled into stage V in wild-type and por1D mitochondria were quantified from three independent import experiments using Multi Gauge software (FujiFilm). The formation of stage V in wild-type mitochondria after 20 min chase was set to 100%. Error bars represent the standard error of the mean.

e6 Molecular Cell 73, 1–10.e1–e7, March 7, 2019

Please cite this article in press as: Ellenrieder et al., Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.014

In Figure 4D, levels of [35S]AAC co-purified with Por1HA or the wild-type control (elution fraction) were quantified using Multi Gauge software (FujiFilm) and normalized to the amount of total [35S]AAC imported (load fraction). The amount of co-purified [35S]AAC arrested at stage III via Por1HA was set to 100%. Error bars represent the standard error of the mean from three independent experiments. In Figure S2A, the membrane potential of wild-type and por1D mitochondria was relatively assessed by calculating the difference in fluorescence before and after addition of valinomycin. The membrane potential of wild-type mitochondria was set to 100%. Error bars represent the standard error of the mean from three independent experiments. In Figure S2D, levels of main phospholipid classes were quantified using Multi Gauge software (FujiFilm) and normalized to the amount of total phospholipids applied. The amounts of the indicated phospholipid classes in wild-type mitochondria were set to 100%. Error bars represent the standard error of the mean from three independent experiments. In Figure S2F, levels of Tim9, Tim10 and Tim12 were quantified using Multi Gauge software (FujiFilm) and normalized to the amount of total protein applied. The protein levels in wild-type mitochondria were set to 100%. Error bars represent the standard error of the mean from three independent experiments. In Figure S3C, the amounts of [35S]AAC assembled into stage V in wild-type and por1 mutant mitochondria were quantified from five independent import experiments using Multi Gauge software (FujiFilm). The formation of stage V in wild-type mitochondria after 12 min chase was set to 100%. Error bars represent the standard error of the mean. In Figure S4A, levels of [35S]AAC co-purified with antibodies against Tim10 from wild-type and por1D mitochondria (elution fraction) were quantified using Multi Gauge software (FujiFilm) and normalized to the amount of total [35S]AAC imported (load fraction). The amount of co-purified [35S]AAC from wild-type mitochondria was set to 100%. Error bars represent the standard error of the mean from three independent experiments.

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