Biogenesis of mitochondrial outer membrane proteins, problems and diseases.

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Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2015-0170 Review

Biogenesis of mitochondrial outer membrane proteins, problems and diseases Lars Ellenrieder1,2, Christoph U. Mårtensson1,2 and Thomas Becker1,3,*

1

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany

2

Fakultät für Biologie, Universität Freiburg, 79104 Freiburg, Germany

3

BIOSS Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany

*Corresponding author e-mail: [email protected]

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L. Ellenrieder et al.: Mitochondrial protein biogenesis

Abstract Proteins of the mitochondrial outer membrane are synthesized as precursors on cytosolic ribosomes and sorted via internal targeting sequences to mitochondria. Two different types of integral outer membrane proteins exist: proteins with a transmembrane β-barrel and proteins embedded by a single or multiple α-helices. The import pathways of these two types of membrane proteins differ fundamentally. Precursors of β-barrel proteins are first imported across the outer membrane via the translocase of the outer membrane (TOM complex). The TOM complex is coupled to the sorting and assembly machinery (SAM complex), which catalyzes folding and membrane insertion of these precursors. The mitochondrial import machinery (MIM complex) promotes import of proteins with multiple α-helical membrane spans. Depending on the topology precursors of proteins with a single α-helical membrane anchor are imported via several distinct routes. We summarize current models and open questions of biogenesis of mitochondrial outer membrane proteins and discuss the impact of malfunctions of protein sorting on the development of diseases.

Keywords: MIM complex; mitochondria; outer membrane; protein import; SAM complex; TOM complex.

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Introduction Mitochondria originated from the engulfment of a α-proteobacterium related prokaryote by a eukaryotic ancestor cell. During the course of evolution the vast majority of the genetic information of the endosymbiont was transferred to the host genome. Eventually, the prokaryotic progenitor cell was transformed to a cell organelle. Mitochondria fulfill essential functions for cellular metabolism like ATP production, synthesis of amino acids, phospholipids, heme and iron-sulfur clusters. Mitochondrial dysfunction and impaired biogenesis lead to a number of severe diseases in particular of neurodegenerative disorders (Rugarli and Langer, 2012; Nunnari and Suomalainen, 2012). Yeast and human mitochondria contain about 1000 and 1500 proteins, respectively (Sickmann et al., 2003; Reinders et al., 2006; Pagliarini et al., 2008). Although mitochondria still contain their own DNA, 99% of the mitochondrial proteins are nuclear-encoded and synthesized on cytosolic ribosomes as precursors. All these precursor proteins are imported into mitochondrial subcompartments by dedicated protein translocases (Neupert and Herrmann, 2007; Endo and Yamano, 2009; Hewitt et al., 2011; Becker et al., 2012). Due to their endosymbiotic origin two membranes, the outer and inner membrane, surround mitochondria. Both membranes confine two aqueous compartments, the intermembrane space and the innermost mitochondrial matrix. The outer membrane forms the border between mitochondria and the cytosol. Outer membrane proteins mediate apoptosis, mitophagy, mitochondrial fusion and fission and tether the cell organelle to other cellular membranes like the endoplasmic reticulum (ER) (Martinou and Youle, 2011; Friedman and Nunnari, 2014; Klecker et al., 2014; Pickrell and Youle, 2015). They enable the communication between mitochondria and the cellular environment. Molecular channels and pores transport precursor proteins and small compounds across the outer membrane. Strikingly, the mitochondrial outer membrane harbors two types of integral membrane proteins: proteins with a single or multiple membrane-spanning α-helices and proteins with a transmembrane β-barrel (Figure 1). All these proteins are synthesized on cytosolic ribosomes and contain internal targeting sequences. Distinct protein machineries evolved to mediate the insertion of these proteins into the outer membrane. Studies of the last few years unraveled a surprising complex set of import pathways and mechanisms for the biogenesis of outer membrane proteins.

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The general entry gate into mitochondria Mitochondrial precursor proteins are delivered by cytosolic factors to the organelle surface. The translocase of the outer membrane (TOM complex) forms the entry gate for most of the mitochondrial proteins. Single particle studies of isolated TOM complexes revealed the existence of two to three pores (Künkele et al., 1998; Ahting et al., 1999; Model et al., 2002). The central component of the TOM complex is Tom40, which forms the protein-conducting channel (Hill et al., 1998; Ahting et al., 1999; Suzuki et al., 2004). This β-barrel protein associates with six proteins, which are integrated into the membrane via a single transmembrane α-helix: the receptor proteins Tom20, Tom22 and Tom70 and the small Tom proteins Tom5, Tom6 and Tom7. Although Tom20 and Tom70 have partly overlapping functions both receptors bind preferentially different types of precursor proteins. Tom20 recognizes precursor proteins with a cleavable presequence, while hydrophobic precursors like carrier proteins are guided by molecular chaperones to the Tom70 receptor (Brix et al., 1997; Young et al., 2003; Yamano et al., 2008). Tom22 is a multifunctional protein exposing a soluble domain towards the cytosol and the intermembrane space. The cytosolic domain of Tom22 acts together with Tom20 in preprotein recognition and provides a docking site for Tom20 and Tom70 (van Wilpe et al., 1999; Yamano et al., 2008; Shiota et al., 2011). The intermembrane space exposed domain of Tom22 is involved in the sorting of precursors to other protein translocases (Shiota et al., 2011). The small Tom proteins Tom5, Tom6 and Tom7 are involved in precursor transfer and regulate assembly and stability of the TOM complex. Tom5 and Tom6 promote formation of the TOM complex, while Tom7 destabilizes the translocase (Alconada et al., 1995; Hönlinger et al., 1996; Dietmeier et al., 1997; Esaki et al. 2004; Schmitt et al., 2005; Sherman et al., 2005; Dukanovic et al., 2009; Becker et al., 2010; Yamano et al., 2010a; Becker et al., 2011a).

Distinct pathways for precursor proteins into mitochondria After passage through the TOM channel dedicated protein machineries sort the precursor proteins into the inner mitochondrial subcompartments (Figure 2) (Neupert and Herrmann, 2007; Endo and Yamano, 2009; Hewitt et al., 2011; Becker et al., 2012). The presequence translocase (TIM23 complex) transports precursor proteins with a cleavable presequence in a membrane potential-dependent manner into and across the inner membrane (Chacinska et al., 2005; Neupert and Herrmann, 2007; Endo and Yamano, 2009; Malhotra et al., 2013). The 4 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis TIM23 complex interacts dynamically with the presequence translocase associated motor (PAM) to promote protein transport into the matrix. The central component of the PAM module, the mitochondrial Hsp70, completes the import of precursor proteins into the matrix upon ATP hydrolysis (Liu et al., 2003; Neupert and Herrmann, 2007, Hewitt et al., 2011). The presequence is removed by the mitochondrial processing peptidase (MPP). The hydrophobic precursors of carrier proteins are transported by small Tim chaperones through the intermembrane space to the carrier translocase (TIM22 complex), which mediates the membrane potential-dependent integration of these precursors into the inner membrane (Koehler et al., 1998; Sirrenberg et al., 1998; Rehling et al., 2003). Mitochondrially-encoded proteins are co-translationally inserted into the inner membrane via the oxidase assembly (OXA) machinery (Hell et al., 2001; Szyrach et al., 2003). The core subunit of the mitochondrial intermembrane space import and assembly machinery Mia40 promotes import and oxidative folding of intermembrane space proteins, which contain a conserved cysteine-rich motif. The electrons are transferred via Erv1 to the respiratory chain to regenerate Mia40 for another cycle of import (Chacinska et al., 2004; Mesecke et al., 2005; Kawano et al., 2009). In addition, two common import pathways into the outer membrane have been found as described below: proteins with a transmembrane β-barrel are inserted via the sorting and assembly machinery (SAM complex), while proteins with multiple α-helical membrane spans are integrated by the mitochondrial import (MIM) machinery (Figure 2). Altogether, seven protein sorting machineries form a dynamic network to transport precursor proteins into the mitochondrial subcompartments.

Biogenesis of outer membrane proteins with α-helical membrane anchor Targeting to mitochondria Outer membrane proteins are inserted into the outer membrane by single or multiple α-helical membrane anchors. Based on the location of the transmembrane domain (TMD) single-spanning

proteins

are

further

divided

into

proteins

with

an

N-terminal

(signal-anchored), internal (internally-anchored) or C-terminal (tail-anchored) membrane span (Figure 1). The targeting information resides within the TMD and positively charged residues in the flanking regions (Kanaji et al., 2000; Horie et al., 2002; Beilharz et al., 2003; Ahting et al., 2005). How specific transport of outer membrane precursor proteins from the 5 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis ribosomes to their target membrane is warranted is poorly understood. Recently, the cytosolic J-domain containing protein Djp1 was shown to specifically mediate mitochondrial localization of a central subunit of the MIM complex, Mim1 (Papic et al., 2013). Interestingly, targeting of tail-anchored proteins to the endoplasmic reticulum (ER) and to mitochondria seems to be closely connected. The ER-localized tail-anchored proteins are imported via the guided entry of tail-anchored proteins (GET) pathway (Hegde and Keenan, 2011; Borgese and Fasana, 2011). In this pathway, the TMD recognition complex (TRC complex) binds to the membrane span of the client protein after its release from the ribosome. The TRC complex transfers precursors to Get3 for transport to the ER membrane. In yeast, Sgt2 of the TRC complex binds strongly to the transmembrane segment of ER proteins, while the mitochondrial Fis1 is recognized with low efficiency (Wang et al., 2010). However, introduction of additional hydrophobic residues in the TMD of Fis1 leads to efficient recognition by Sgt2 and insertion of the mutant form of Fis1 into the ER membrane (Wang et al., 2010). Moreover, upon disruption of the GET pathway ER-resident proteins mislocalize to mitochondria (Schuldiner et al., 2008) and are removed by the AAA-ATPase Msp1 (Okreglak and Walter, 2014; Chen et al., 2014). Thus, the TRC complex may act as an important control point to sort tail-anchored proteins either to the ER or to mitochondria. Despite the link to the GET pathway, no specific factors that target tail-anchored proteins to mitochondria has been identified so far. Sorting into the outer membrane of mitochondria The MIM machinery plays a central role in the biogenesis of various α-helically embedded outer membrane proteins. Tom70 recognizes precursors of multi-spanning outer membrane proteins and transfers them to the MIM machinery, which mediates their membrane integration and assembly (Figure 3A) (Otera et al., 2007; Becker et al., 2011b; Papic et al., 2011). The MIM complex further promotes the biogenesis of the signal-anchored Tom20 and Tom70, which expose a large soluble domain into the cytosol (Figure 3B) (Becker et al., 2008; Hulett et al., 2008; Popov-Celeketić et al., 2008; Dimmer et al., 2012). It was also observed that insertion and assembly of the tail-anchored small Tom proteins are reduced in the absence of a functional MIM complex (Becker et al., 2008; Thornton et al., 2010). Thus, the MIM machinery promotes the biogenesis of proteins with different topology. Insights into structure and mechanism of the MIM complex are limited. For instances, it is not known whether the MIM machinery acts as an insertase for these outer membrane proteins and how it copes with different types of substrates. The MIM complex is composed of Mim1 6 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis (also termed Tom13) and Mim2, which are both crucial for function and stability of the protein complex (Ishikawa et al., 2004; Waizenegger et al., 2005; Becker et al., 2008; Popov-Celeketić et al., 2008; Hulett et al., 2008; Becker et al., 2011b; Papic et al., 2011; Dimmer et al., 2012). Interestingly, Mim1 action depends on the formation of oligomers (Popov-Celeketić et al., 2008). Future studies have to reveal whether the Mim1 oligomer provides a suitable environment to bind and insert outer membrane proteins. Homologous proteins of Mim1 and Mim2 were found in fungi, but were not identified in higher eukaryotes (Dimmer and Rapaport, 2010; Dimmer et al., 2012). Whether a specific protein sorting machinery for α-helically embedded outer membrane proteins exists in mammalian mitochondria is unknown. Several additional import pathways were reported for single-spanning proteins. Om45 is an abundant signal-anchored outer membrane protein in yeast that exposes a large domain into the intermembrane space (Lauffer et al., 2012; Wenz et al., 2014; Song et al., 2014). Recent studies revealed that the precursor of Om45 is transported across the outer membrane via the TOM complex. Strikingly, the TIM23 complex of the inner membrane is additionally required to complete the translocation of the soluble domain of Om45 (Wenz et al., 2014; Song et al., 2014). The final assembly of Om45 into the outer membrane depends on the presence of the MIM complex (Figure 3C) (Wenz et al., 2014). Taz1 has a similar topology like Om45 and is also first imported via the TOM complex and then transported by the small Tim proteins to the outer membrane (Brandner et al., 2005; Herndon et al., 2013). Thus, the TOM complex mediates the transport of outer membrane proteins with soluble domains exposed to the intermembrane space. Further reports indicate a broader role of TOM subunits in the import of single-spanning outer membrane proteins (Figure 3D). First, the import of the Tom20 precursor is affected by blocking Tom40 (Schneider et al., 1991; Ahting et al., 2005). Since Tom20 is also a component of the TOM complex, indirect effects cannot be excluded. Second, conflicting results were reported whether or not TOM subunits are involved in the import of the tail-anchored Bax (Bellot et al., 2007; Ott et al., 2007; Sanjuán Sklarz et al., 2007). Third, it was proposed that the TOM complex might mediate lateral release of proteins into the outer membrane (Harner et al., 2011a). In the same line accumulation of the PTEN-induced putative kinase (PINK1) at the mitochondrial surface of depolarized human mitochondria involves the TOM complex (Lazarou et al., 2012). It was reported that PINK1 is laterally released into the outer membrane by the TOM complex in a Tom7-dependent manner (Figure 3D) (Hasson et al., 2013; Pickrell and Youle, 2015). More experimental data

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L. Ellenrieder et al.: Mitochondrial protein biogenesis are needed to define the role of the TOM complex in the biogenesis of outer membrane proteins with a α-helical membrane span. Furthermore, the SAM complex is required for the insertion and assembly of the Tom22 precursor that contains an internal membrane anchor (Stojanovski et al., 2007; Dukanovic et al., 2009; Lackey et al., 2011). After initial recognition by TOM receptors the Tom22 precursor engages a subpopulation of the SAM complex, which additionally contains the mitochondrial division and morphology protein Mdm10 (Figure 3E) (Keil and Pfanner, 1993; Meisinger et al., 2004; Thornton et al., 2010; Becker et al., 2011a). Finally, the import of tail-anchored proteins like Fis1 and Mcr1 into the outer membrane seems to occur independently of known protein translocases (Figure 3F) (Setoguchi et al., 2006; Kemper et al., 2008; Meineke et al., 2008). Instead, the low sterol content in the outer membrane was found to be important for the membrane-integration of Fis1 (Kemper et al., 2008; Krumpe et al., 2012). Thus, a common import pathway has been identified for multi-spanning outer membrane proteins, whereas the single-spanning proteins embark a variety of import pathways. The molecular mechanisms of membrane insertion and folding have to be investigated to understand why so many different translocases are involved in the biogenesis of single-spanning outer membrane proteins.

Biogenesis of mitochondrial β-barrel proteins Transport into the outer membrane Proteins with a transmembrane β-barrel were only found in the outer membrane of Gram-negative bacteria and of cell organelles with endosymbiotic origin like plastids of plant cells and mitochondria. How precursors of the β-barrel proteins are targeted to the mitochondrial surface is not understood. So far, no consensus sequence was identified that directs the precursors to mitochondrial protein receptors. Interestingly, bacterial and plastid β-barrel proteins expressed in yeast are also transported to mitochondria (Walther et al., 2009; Ulrich et al., 2012; Ulrich et al., 2014) indicating that a structural feature rather than a conserved sequence motif is important. Furthermore, these observations reveal that key elements of the biogenesis pathway of β-barrel precursors are conserved from bacteria to eukaryotes. Tom20 and Tom70 of the TOM complex recognize the precursors of β-barrel proteins on the mitochondrial surface (Yamano et al., 2008; Krimmer et al., 2001; Habib et al., 2005;

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L. Ellenrieder et al.: Mitochondrial protein biogenesis Kozjak-Pavlovic et al., 2007). Subsequently, the precursors are transported via the Tom40 channel to the intermembrane space (Paschen et al., 2003; Wiedemann et al., 2003; Humphries et al., 2005). Strikingly, the TOM complex is coupled via Tom22 to the SAM complex, which mediates folding and membrane integration of the β-barrel precursor (Figure 4A) (Qiu et al., 2013). The transfer step is assisted by the small Tim proteins that bind to the TOM-SAM associated precursor. Loss of the TOM-SAM supercomplex or the small Tim proteins impairs folding and assembly of β-barrel precursors (Hoppins and Nargang, 2004; Wiedemann et al., 2004; Qiu et al., 2013). The SAM complex recognizes incoming β-barrel precursors via a conserved β-signal, which is located within the last β-strand (Kutik et al., 2008). The SAM complex is composed of three subunits. The β-barrel protein Sam50 (also referred to as Tob55/Omp85) is associated with two peripheral proteins Sam35 (Tob38, Tom38) and Sam37 (Mas37, Tom37), which are both exposed to the cytosol (Wiedemann et al., 2003; Paschen et al., 2003; Kozjak et al., 2003; Gentle et al., 2004; Milenkovic et al., 2004; Ishikawa et al., 2004; Waizenegger et al., 2004; Lackey et al., 2011). The three SAM subunits are associated in a 1:1:1 stoichiometry within the SAM complex (Klein et al., 2012). Two of the subunits, Sam35 and Sam50 are essential for life (Kozjak et al., 2003; Paschen et al., 2003; Milenkovic et al., 2004; Waizenegger et al., 2004; Ishikawa et al., 2004), indicating the central role of this pathway for mitochondrial biogenesis. The function of the individual components is poorly understood. Sam35 recognizes the β-signal of the substrate protein, whereas Sam37 is involved in the release of the precursor from the SAM complex (Chan and Lithgow, 2008; Kutik et al., 2008). The β-barrel protein Sam50 belongs to the Omp85 protein family, which is conserved from bacteria to humans (Gentle et al., 2004). It contains a β-barrel and a single polypeptidetransport-associated (POTRA) domain, which is exposed to the intermembrane space (Habib et al., 2007; Kutik et al., 2008; Qiu et al., 2013). Deletion of the POTRA domain of Sam50 leads to impaired release of the β-barrel precursor into the outer membrane (Stroud et al., 2011). The β-barrel of Sam50 exhibits channel activity and is crucial for the integration and folding of the precursor (Paschen et al., 2003; Bredemeier et al., 2007; Kutik et al., 2008; Qiu et al., 2013). The molecular mechanisms of the Sam50 activity are not understood. Indications how Sam50 might operate derive from crystal structures of the bacterial homolog BamA (Noinaj et al., 2013), which mediates the membrane integration of β-barrel proteins in the outer membrane of Gram negative bacteria (Voulhoux et al., 2003; Wu et al., 2005). Strikingly, the binding between the first to the last β-strand appears to be weaker than the association of other β-strands of BamA and could allow the formation of a 9 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis lateral gate within the β-barrel of BamA. One attractive model is that precursors enter the β-barrel of BamA and are released into the membrane via the lateral gate. Alternatively, the precursors might insert at the interface between BamA and the lipid bilayer (Noinaj et al., 2013). Future studies have to shed light on the fascinating molecular mechanism of the SAM complex in mitochondria. The β-barrel pathway is part of a mitochondrial network Recent studies revealed that the import pathway of β-barrel precursors is integrated into a protein network for mitochondrial biogenesis. The SAM complex interacts with components of the mitochondrial contact site and cristae organization system (MICOS) (Figure 4B), which is crucial to maintain the architecture of the inner membrane (Xie et al., 2007; Harner et al., 2011b; Hoppins et al., 2011; von der Malsburg et al., 2011; Bohnert et al., 2012; Zerbes et al., 2012; Körner et al., 2012; Ott et al., 2012; Pfanner et al., 2014). The POTRA domain of Sam50 provides the docking site for the MICOS complex (Bohnert et al., 2012). The function of both protein complexes seems to be closely linked. Reduced levels of Sam50 or Sam35 affect the cristae organization (Körner et al., 2012; Ott et al., 2012), whereas alterations of the levels of Mic60, a core subunits of MICOS, impair the biogenesis of β-barrel proteins (Körner et al., 2012; Bohnert et al., 2012). The β-barrel pathway is also closely connected to the ER-mitochondria encounter structure (ERMES) (Figure 4C), which links mitochondria and the ER in yeast (Kornmann et al., 2009). Deletion of core subunits of the ERMES complex impairs the biogenesis of β-barrel proteins (Meisinger et al., 2007; Wideman et al., 2010; Wideman et al., 2013). However, no direct involvement of the ERMES complex in β-barrel biogenesis was demonstrated so far. In addition, the SAM complex is linked to ERMES on a molecular level: Mdm10 associates with the SAM complex and is also a core subunit of the ERMES complex (Boldogh et al., 2003; Meisinger et al., 2004; Meisinger et al., 2007; Yamano et al., 2010b, Klein et al., 2012; Wideman et al., 2010; Becker et al., 2011a). Binding of Tom7 dissociates Mdm10 from the SAM complex and promotes association of Mdm10 with the ERMES complex (Figure 4C) (Meisinger et al., 2006; Yamano et al., 2010a; Becker et al., 2011a). Why Mdm10 is present at the SAM and ERMES complexes is unknown. Molecular studies on the individual Mdm10 populations will be important to understand the function of the link between SAM and ERMES complexes.

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The assembly of outer membrane protein complexes The formation of several outer membrane protein complexes involves the association of peripheral or single spanning proteins with a transmembrane β-barrel. This feature imposes challenges on the assembly process, which do not apply when protein complexes of other cellular membranes are built up. Intensive studies on the formation of the TOM complex revealed a concerted action of distinct protein translocases (Figure 5). The precursor of the β-barrel protein Tom40 is imported via the TOM-SAM complex. The binding of the small Tom proteins Tom5 and Tom6 to the Tom40 precursor occurs already at the SAM complex (Dukanovic et al., 2009; Becker et al., 2010). In the absence of Mim1 the formation of this intermediate is impaired (Waizenegger et al., 2005; Becker et al., 2008; Becker et al., 2010) indicating that the MIM machinery could deliver the small Tom proteins for assembly with the Tom40 precursor. The SAM-Mdm10 complex is crucial for the formation of the TOM complex. Two possible functions have been proposed. First, the SAM-Mdm10 complex delivers Tom22 to the assembly intermediate that consists of Tom40 and the small Tom proteins to form the TOMcore complex (Meisinger et al., 2004; Stojanovski et al., 2007; Thornton et al., 2010; Becker et al., 2011a). Second, it was reported that binding of Mdm10 to the SAM complex stimulates the release of the Tom40 precursor to allow further assembly with other Tom proteins (Yamano et al., 2010b). Finally, the MIM machinery promotes the association of the receptor proteins Tom20 and Tom70 with the TOMcore complex to form the mature TOM complex (Becker et al., 2008; Hulett et al., 2008; Popov-Celeketic et al., 2008). Interestingly, an excess of free Tom7 negatively regulates the assembly of the TOM complex by blocking the initial assembly stages of Tom40 and dissociating the SAM-Mdm10 complex (Meisinger et al., 2006; Yamano et al., 2010a; Becker et al., 2011b). In conclusion, the formation of the TOM complex is a stepwise process involving the coordinated cooperation of two SAM complexes and the MIM machinery.

The role of dysfunctions of mitochondrial protein import in diseases Studies of the last few years revealed that mitochondrial protein import plays an important role in the development of diseases (Sokol et al., 2014). For instance, carcinogenesis involves the shift of cellular metabolism from respiratory to more glycolytic energy production (Warburg effect). One can speculate that changes in protein sorting occur to adjust mitochondrial function to cellular metabolism in cancer cells. Supporting this idea, several 11 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis subunits of the protein transport machineries like mitochondrial Hsp70 (Mortalin in human), TIMM17A, TIMM50 of the TIM23 complex and MIA40 are upregulated in various cancer cells, which is associated with low survival prognosis (Dundas et al., 2005; Wadhwa et al., 2006; Xu et al., 2010; Sankala et al., 2011; Yang et al., 2012). In an alternative model, the upregulation of protein import components promotes the transport of the transcription factor p53 into mitochondria. The mitochondrially-localized p53 cannot induce apoptosis, which in turn favors growth of the tumor cells (Lu et al., 2011; Sokol et al., 2014). Mitochondrial dysfunctions are often linked to neurodegenerative disorders (Rugarli and Langer, 2012; Nunnari and Suomalainen, 2012). Several diseases of the nervous systems has been linked to defects in mitochondrial protein sorting. First, in mitochondria from patients suffering Alzheimer´s disease the amyloid precursor accumulates in a TOM-TIM23 supercomplex, which impairs the import of mitochondrial precursors (Devi et al., 2006). Furthermore, mitochondrially-localized amyloid β-peptides block the processing of imported precursors (Mossmann et al., 2014). Second, mutant forms of the superoxide dismutase (SOD1), which cause amyotrophic lateral sclerosis (ALS), accumulate uncontrolled at the mitochondrial surface and intermembrane space (Cozzolino et al., 2013). The protein import is delayed in mitochondria isolated from spinal cord of transgenic rats expressing the pathogenic mutant form of SOD1 (Li et al., 2010). Third, the PINK1 and Parkin mediated quality control system of mitochondria is affected in patients suffering Parkinson´s disease (Pickrell and Youle, 2015). Protein sorting plays a fundamental role in this process. In healthy mitochondria PINK1 is imported into the inner membrane and subsequently degraded. When the membrane potential is depleted PINK1 accumulates at the outer membrane in a TOM complex-dependent manner (Lazarou et al., 2012; Hasson et al., 2013; Okatsu et al., 2015). Subsequently, outer membrane localized PINK1 recruits and activates the E3 ubiquitin ligase Parkin, which in turn labels outer membrane proteins by ubiquitination for proteasomal degradation and thus promotes mitophagy (Pickrell and Youle, 2015). The outer membrane protein translocases form also an entry gate for pathogenic effector proteins from viruses or bacteria to affect mitochondrial function (Jiang et al., 2011; Yoshizumi et al., 2014). Interestingly, upon infection with Neisseria gonorrhoeae the bacterial β-barrel protein PorB is imported into mitochondria following the TOM-SAMdependent import pathway of β-barrel precursors (Jiang et al., 2011). Whether or not the mitochondrially-localized PorB sensitizes to or protects cells from apoptosis is not clear

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L. Ellenrieder et al.: Mitochondrial protein biogenesis (Massari et al., 2003; Kozjak-Pavlovic et al., 2009). Thus, pathogenic effector proteins can enter mitochondria embarking established import mechanisms.

Perspectives Biogenesis of outer membrane proteins seems to involve a complex set of cytosolic and membrane-bound protein factors. How the transport through the cytosol to the target organelle is accomplished is unclear. Since specific cytosolic targeting systems for ER proteins have been identified the presence of similar sorting factors for mitochondrial proteins is likely. Identification of such systems will help to understand how the biogenesis of cell organelles is coordinated in the cellular context. Whereas cytosolic targeting is largely uncharacterized, two main protein sorting machineries have been identified in the mitochondrial outer membrane. The SAM complex mediates the biogenesis of β-barrel precursors, whereas the MIM complex is important for the import of α-helically embedded proteins. Insights into the molecular mechanisms of protein insertion in particular by the MIM machinery are limited. Open questions concern the composition and function of the MIM complex. It is not clear how the MIM machinery recognizes its substrates and whether it acts as an insertase. Studies on the interaction partners of the SAM complex indicated that protein transport is part of a protein network for mitochondrial biogenesis. The functional implications of the integration of protein biogenesis into a protein network for mitochondrial architecture and function are not entirely understood. One possibility is that protein import can be adjusted to the physiological state of the mitochondria. The emerging role of protein biogenesis in diseases revealed the importance of understanding the molecular processes of outer membrane protein biogenesis for human health.

Acknowledgements: We thank Dr. Lukasz Opaliński for discussion. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 746, BE4679/2-1 and the Excellence Initiative of the German Federal and State Governments (EXC 294 BIOSS Centre for Biological Signalling Studies).

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L. Ellenrieder et al.: Mitochondrial protein biogenesis Yamano, K., Tanaka-Yamano, S., and Endo, T. (2010b). Mdm10 as a dynamic constituent of the TOB/SAM complex directs coordinated assembly of Tom40. EMBO Rep. 11, 187193. Yang, J., Staples, O., Thomas, L.W., Briston, T., Robson, M., Poon, E., Simões, M.L., ElEmir, E., Buffa, F.M., Ahmed, A., et al. (2012). Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression. J. Clin. Invest. 122, 600-611. Yoshizumi, T., Ichinohe, T., Sasaki, O., Otera, H., Kawabata, S.-I., Mihara, K., and Koshiba, T. (2014). Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity. Nat. Commun. 5, 4713. Young, J.C., Hoogenraad, N.J., and Hartl, F.U. (2003). Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell 112, 41-50. Zerbes, R.M., Bohnert, M., Stroud, D.A., von der Malsburg, K., Kram, A., Oeljeklaus, S., Warscheid, B., Becker, T., Wiedemann, N., Veenhuis, M., et al. (2012). Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains. J. Mol. Biol. 422, 183-191.

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Tables and figures

Figure 1:

Different types of mitochondrial outer membrane proteins.

Integral outer membrane proteins are anchored either by a transmembrane β-barrel or by single or multiple α-helices. The single-spanning proteins are further distinguished according to the localization of the transmembrane segment into signal-anchored (N-terminal), internally-anchored and tail-anchored (C-terminal) proteins.

Figure 2:

Protein import pathways into mitochondria.

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L. Ellenrieder et al.: Mitochondrial protein biogenesis The translocase of the outer membrane (TOM complex) forms the entry gate for most mitochondrial precursor proteins. After passage through the TOM channel several different sorting pathways branch off. Precursors with a cleavable presequence are transferred to the presequence translocase (TIM23 complex), which sorts the precursor in a membrane potential (Δψ)-dependent manner into the inner membrane or into the mitochondrial matrix. The latter import process depends additionally on the ATP-dependent activity of the presequence translocase associated motor (PAM). The presequence is removed by the mitochondrial processing peptidase (MPP). Precursors of carrier proteins are guided through the intermembrane space by the small Tim proteins to the carrier translocase (TIM22 complex), which mediates their membrane potential-dependent integration into the inner membrane. The oxidase

assembly

(OXA)

machinery

inserts

mitochondrially-encoded

proteins

co-translationally into the inner membrane. The mitochondrial intermembrane space import and assembly machinery (MIA) mediates import and oxidative folding of proteins containing a cysteine-rich motif. The small Tim proteins transfer precursors of β-barrel proteins to the sorting and assembly machinery (SAM complex), which sorts the precursors into the outer membrane. The mitochondrial import machinery (MIM complex) inserts precursors with multiple α-helical membrane spans into the outer membrane.

Figure 3:

Models for import of precursor of α-helically embedded mitochondrial outer

membrane proteins. (A) Precursors of multi-spanning proteins are recognized by Tom70 and transferred to the MIM complex, which is crucial for their assembly into the outer membrane. (B) The MIM complex mediates the biogenesis of precursors of the signal-anchored Tom20 and Tom70. (C) The precursor of Om45 is imported via the TOM channel with the help of the TIM23 complex 26 / 29


L. Ellenrieder et al.: Mitochondrial protein biogenesis into the intermembrane space. The MIM complex promotes Om45 assembly into the outer membrane. (D) TOM subunits promote the biogenesis of several single-spanning outer membrane proteins. (E) The precursor of Tom22 is recognized by TOM receptors and inserted into the outer membrane by the SAM-Mdm10 complex. (F) The membrane integration of the Fis1 and Mcr1 precursors occur independently of proteins.

Figure 4:

Biogenesis of β-barrel proteins.

(A) Precursors of β-barrel proteins are imported via the TOM channel and transferred with the help of the small Tim proteins to the SAM complex. The SAM complex mediates membrane integration and folding of the precursors. The TOM and SAM complexes are coupled via

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L. Ellenrieder et al.: Mitochondrial protein biogenesis Tom22 to coordinate import and insertion. (B) The TOM and SAM complexes associate with Mic60 of the mitochondrial contact site and cristae organizing system (MICOS). (C) The SAM complex is linked via Mdm10 to the ER-mitochondria encounter structure (ERMES). Mdm10 binds the SAM complex and is a component of the ERMES complex. Tom7 interacts with Mdm10 and induces dissociation of the SAM-Mdm10 complex.

Figure 5:

Model for the multi-step assembly of the TOM complex.

The precursor of Tom40 is imported via the TOM-SAM pathway (not depicted). Small Tom proteins bind to the SAM-bound Tom40 precursor. The MIM machinery provides the small Tom proteins for the assembly steps. The SAM-Mdm10 complex mediates assembly of the Tom22 precursor with the Tom40/small Tom proteins intermediate to form the TOMcore complex. Finally, the MIM machinery promotes assembly of the peripheral receptors Tom20 and Tom70 with the TOMcore complex.

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Author’s corner

Lars Ellenrieder studied Molecular Medicine at the University of Freiburg. Currently, he is a PhD student in the group of Thomas Becker studying protein biogenesis and protein complex formation of the mitochondrial outer membrane.

Christoph U. Mårtensson studied Molecular Life Science at the University of Lübeck and was Erasmus student at the Imperial College London. Currently, he is a PhD student in the group of Thomas Becker and is working on the characterization of novel interaction partners of the TOM and MIM complexes.

Thomas Becker, PhD, received his training at the Universities of Kiel, Munich and Freiburg. Since 2009 he is group leader (Assistant Professor) at the University of Freiburg. Research areas: mitochondrial protein sorting, protein networks and cytosolic protein targeting.

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Outer Membrane Biogenesis.

Periplasmic quality control in biogenesis of outer membrane proteins.

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

Assembly of β-barrel proteins in the mitochondrial outer membrane.

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria.

The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1.

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

Outer membrane proteins of Pseudomonas.

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

Biogenesis of mitochondrial proteins in HeLa cells.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis.

Ionic permeability of the mitochondrial outer membrane.

Outer membrane protein biogenesis in Gram-negative bacteria.

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The deadly landscape of pro-apoptotic BCL-2 proteins in the outer mitochondrial membrane.

Bak through the outer mitochondrial membrane.

Biogenesis and folding of β-barrel membrane proteins.

Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets.

Identification of FkpA as a key quality control factor for the biogenesis of outer membrane proteins under heat shock conditions.

Mitochondrial outer membrane permeabilization: a focus on the role of mitochondrial membrane structural organization.

Misfolded proteins, mitochondrial dysfunction, and neurodegenerative diseases.