Import of ribosomal proteins into yeast mitochondria.

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Biochemistry and Cell Biology Invited review article

Import of ribosomal proteins into yeast mitochondria Michael W. Woellhaf1, Katja G. Hansen1, Christoph Garth2, Johannes M. Herrmann1*

1, Cell Biology, University of Kaiserslautern, Germany 2, Computational Topology, University of Kaiserslautern, Germany

*corresponding author Johannes Herrmann, Cell Biology, University of Kaiserslautern Erwin-Schrödinger-Strasse 13, 67663 Kaiserslautern, Germany phone +49 631 2052406, fax +49 631 2052492, email: [email protected]

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Abstract Mitochondrial ribosomes of baker’s yeast contain at least 78 protein subunits. All but one of these proteins are nuclear encoded, synthesized on cytosolic ribosomes and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and showed indeed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix and thereby specifically focus on proteins of the mitochondrial ribosome.

Key words Mitochondria; Protein Translocation; Targeting Sequences; Ribosomes

Abbreviations IMS, intermembrane space; MPP, matrix processing peptidase; MRP, mitochondrial ribosomal protein; MTS, mitochondrial targeting sequence; pI, isoelectric point; TIM, translocase of the inner membrane of mitochondria; TOM, translocase of the outer membrane of mitochondria

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The mitochondrial proteome The mitochondrial proteome is very complex. Out of the 5820 yeast proteins listed in the Saccharomyces Genome Database, the MITOP reference set describes 601 as mitochondrial proteins (Elstner et al. 2009) and this list is steadily increasing. Mammalian mitochondria contain even more proteins and here, the patterns of mitochondrial proteins depend considerably on the specific tissues analyzed (Forner et al. 2006; Pagliarini et al. 2008; Rhee et al. 2013). Many mitochondrial proteins are phylogenetically related to bacterial proteins, nicely showing the prokaryotic origin of mitochondria. However, a large number of proteins were added during evolution of eukaryotic cells. This is obvious from the composition of several mitochondrial protein complexes: they often share a conserved catalytically active core with bacterial enzymes to which a number of mitochondrion-specific peripheral subunits are associated to contribute to complex stability or regulation (Zhang and Broughton 2013). For example, one of the largest matrix complexes, the mitochondrial ribosome, shares many subunits with bacterial ribosomes. However, more than half of all subunits are mitochondrionspecific and were added during the evolution of the eukaryotic cell (Smits et al. 2007). Most of these mitochondrion-specific mitochondrial ribosomal proteins (MRPs) are located on the ribosomal surface whereas many bacteria-like MRPs and the rRNA molecules make up most of the core of the ribosome (Greber et al. 2013; Sharma et al. 2003). Thus, the mitochondrial proteome represents a mosaic of ancestral components (that are related to bacterial proteins) and components of eukaryotic origin, for which no homologs in prokaryotes are found. The mitochondrial DNA was severely reduced during evolution and out of the many hundreds of mitochondrial proteins, only a very small number (e.g. 8 in yeast, 13 in humans) are synthesized on mitochondrial ribosomes (Ott and Herrmann 2010; Rorbach et al. 2007). All other proteins are synthesized in the cytosol from where they are imported into mitochondria.

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Protein import into mitochondria During the last three decades, the processes by which nuclear encoded precursor proteins are imported into mitochondria and sorted into their specific mitochondrial subcompartment were analyzed extensively. A number of comprehensive reviews were published that provide an excellent overview about the mechanisms and constituents of the mitochondrial protein import system (Chacinska et al. 2009; Dudek et al. 2013; Endo et al. 2011; Neupert and Herrmann 2007). Here we will only give a short introduction into the general import pathway that directs preproteins from the cytosol into the matrix.

Matrix proteins contain N-terminal matrix-targeting sequences Matrix-destined proteins contain N-terminal targeting signals that, in most cases, are removed after translocation into the matrix. These matrix-targeting sequences (MTSs, presequences) consist of 15 to 80 amino residues forming amphiphatic helices with one positively charged and one hydrophobic surface (von Heijne 1986). These signals are both necessary and sufficient to direct proteins into mitochondria. The specific properties of MTSs allows the generation of useful algorithms to predict mitochondria targeting probabilities reliably such as TargetP or MitoProt II (Emanuelsson et al. 2007; Guda et al. 2004; Reichert and Neupert 2004). Nuclear-encoded proteins are synthesized on cytosolic ribosomes and maintained in a soluble transport-competent state by cytosolic chaperones (Fig. 1, step1). Translocation across both mitochondrial membranes into the matrix is carried out in two sequential, though functionally and kinetically coupled reactions (Chacinska et al. 2003; Sirrenberg et al. 1997). First, proteins are threaded through the TOM complex in the outer membrane before they are

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passed on to the TIM23 complex in the inner membrane which completes translocation into the matrix.

- Fig 1 -

Translocation across the outer membrane The Translocase of the Outer membrane of Mitochondria (TOM complex) is the main mitochondrial entrance gate. It plays a central role in mitochondrial preprotein import. It contains three cytosol-oriented receptor subunits, Tom70, Tom20 and Tom22, which have high affinity to MTSs and cooperate to recruit preproteins to the mitochondrial surface (Fig. 1, step 2) (Brix et al. 2000; Fan et al. 2011; Hönlinger et al. 1995; Kiebler et al. 1993; Shiota et al. 2011; Söllner et al. 1989; Yamano et al. 2008b). They pass the presequences on to the import pore in the TOM complex (Fig. 1, step 3) that is formed by the membrane-embedded subunits Tom40, Tom5, Tom6 and Tom7 (Ahting et al. 2001; Hill et al. 1998). Tom40 is a βbarrel protein, however, it is unclear whether the pore is formed by the central opening of the β-barrel or between several Tom40 subunits in the TOM complex. Tom5, Tom6 and Tom7 are small subunits of the TOM complex that all contain one single transmembrane span. They are not essential but deletion mutants show defects in the assembly and/or stability of the TOM complex (Becker et al. 2011; Dembowski et al. 2001; Model et al. 2001; Yamano et al. 2010). Current studies implicate that cytosolic kinases regulate protein import at the level of the TOM complex (Opalinska and Meisinger 2014). Several kinases phosphorylate a number of different serine and threonine residues of the TOM subunits which influences their assembly 5


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into the TOM complex or their affinity to precursor proteins (Gerbeth et al. 2013; Rao et al. 2011; Rao et al. 2012; Schmidt et al. 2011). Thereby, cells might adapt the levels of protein import to their metabolic needs.

Translocation across the inner membrane The TIM23 (for Translocase of the Inner Membrane of Mitochondria) complex consists of two parts: a pore-forming subcomplex that is embedded into the inner membrane and the matrix-located import motor which is associated to the membrane-embedded subcomplex. The membrane-embedded part is made up by three essential components (Tim17, Tim23, and Tim50) and one non-essential subunit (Tim21). Tim23 is most likely the pore-forming subunit of the TIM23 complex (Alder et al. 2008; Malhotra et al. 2013; Pareek et al. 2013; Truscott et al. 2001). Upon reconstitution in lipid bilayers, Tim23 shows an ion-conducting capacity in electrophysiological measurements (Martinez-Caballero et al. 2007; Meinecke et al. 2006; Truscott et al. 2001). The TIM23 complex is specifically regulated by preproteins and by the membrane potential (Marom et al. 2011; Zhang and Broughton 2013). Several binding sites for presequences in the intermembrane space (IMS) were identified: initially, preproteins associate with the trans binding site on Tom22 from where they are passed on to the receptor domain of Tim50 (Lytovchenko et al. 2013; Marom et al. 2011; Mokranjac et al. 2009; Schulz et al. 2011; Shiota et al. 2011; Zhang et al. 2012). Tim50, in a concerted function with the IMS domain of Tim23, then directs presequences further into the protein-conducting channel of the TIM23 complex (Donzeau et al. 2000; Geissler et al. 2002; Meinecke et al. 2006; Mokranjac et al. 2003; Yamamoto et al. 2002) (Fig. 1, step 4). Tim17 is structurally and phylogenetically related to Tim23, but its specific function in the import process is not known. It was suggested that Tim17 plays a regulatory function in the gating of the TIM23 complex (Martinez-Caballero et al. 2007; Meier et al. 2005; Pareek et al. 2013). The 6


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membrane-embedded part of the TIM23 translocase is associated with enzymes of the respiratory chain, presumably to improve local membrane potential levels around the import sites (Gebert et al. 2012; Mick et al. 2012; Murcha et al. 2012; van der Laan et al. 2006). The subunits of the import motor are the ATP-regulated matrix chaperone Hsp70, the Hsp70docking protein Tim44, the J-protein Tim14 (Pam18), the J-like protein Tim16 (Pam16) and the non-essential protein Pam17 (D'Silva et al. 2004; D'Silva et al. 2005; Frazier et al. 2004; Kozany et al. 2004; Krayl et al. 2007; Mokranjac et al. 2006; Okamoto et al. 2002; Pais et al. 2011; Yamano et al. 2008a). Hsp70, in its ATP-bound state, binds preproteins during their translocation into the matrix. Thereby substrate binding induces ATP hydrolysis and the release of Hsp70 from Tim44. Repetitive binding reactions of several Hsp70 molecules then drive the complete translocation of preproteins into the matrix (Fig. 1, step 5). Hsp70 together with Hsp60/Hsp10 and other chaperones then support the folding of the imported proteins. During or following translocation into the matrix, most preproteins are proteolytically matured by processing peptidases. The matrix processing peptidase MPP is a dimeric complex that removes the presequences from most matrix-destined proteins (Vögtle et al. 2009). A recently published study on the mitochondrial proteome of yeast identified the N-termini of 615 mature proteins. This study concluded that the vast majority of matrix proteins contains presequences that are removed by MPP. After cleavage by MPP, some proteins are further processed by Icp55 or Oct1 which remove one or eight residues from the N termini, respectively (Naamati et al. 2009; Venne et al. 2013; Vogtle et al. 2011; Vögtle et al. 2009). Many matrix proteins are subunits of oligomeric protein complexes. However, only little is known about the assembly reactions in the matrix, such as the assembly of the mitochondrial ribosome (Fig. 1, step 6).

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Proteins of the mitochondrial ribosome of yeast Mitochondria contain a highly reduced genome that codes only for a very small number of proteins, namely eight in yeast and 13 in mammals including human. Hence, the vast majority of the presumably 3,000-4,000 genes of bacteria that served as mitochondrial ancestors were lost during evolution of eukaryotic cells (Kurland and Andersson 2000). Nevertheless, mitochondrial ribosomes are similarly complex as ribosomes of the bacterial or eukaryotic cytosol. During evolution, the number as well as the lengths of ribosomal proteins increased, even when one subtracts the MTSs (Borst and Grivell 1971; Greber et al. 2013; Neupert 1977; Sharma et al. 2009; Sharma et al. 2003; Smits et al. 2007). This additional protein mass on the ribosome might contribute to the stability, membrane binding or regulation of the mitochondrial translation system and might compensate for a reduction of ribosomal RNA that is seen in a number of organism groups, for example in many animals (Koc et al. 2001a; Koc et al. 2001b; O'Brien 2003; Prestele et al. 2009). A number of proteomic studies identified the protein composition of ribosomes of bovine, human and yeast mitochondria (Gan et al. 2002; Graack et al. 1999; Graack and WittmannLiebold 1998; Gruschke et al. 2010; Koc et al. 2001a; Koc et al. 2001b; Saveanu et al. 2001; Suzuki et al. 2001). In yeast, 78 MRPs were identified so far, 44 in the large (Table 1) and 34 in the small subunit (Table 2). One of these proteins, Var1 the homolog of the E. coli Rps3 protein, is encoded on the mitochondrial genome, all others are encoded by nuclear genes. Most of these proteins are essential for either the stability or the functionality of the mitochondrial translation machinery. 53 of these proteins show similarity to constituents of the E. coli ribosome, and many are related, though not as closely, to proteins of the cytosolic 8


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ribosome. However, there are no dual targeted proteins known which are present both in the mitochondrial and the cytosolic ribosome and the proteomic studies carried out so far suggest that both ribosomes use discrete sets of protein and rRNA constituents. - Table 1 MRPs - large subunit - Table 2 MRPs - small subunit

Most MRPs are basic and smaller than 40 kDa Most MRPs are in a size range of 20 to 40 kDa and on average about 9 kDa larger than proteins of bacterial and cytosolic ribosomes (Fig. 2A). This is due to the presence of MTSs on many of these proteins and of N- and C-terminal size extensions which are found on many MRPs. The role of these mitochondrion-specific extensions is not known. Nevertheless on average, MRPs are smaller than many non-ribosomal matrix proteins which scatter between less than 10 and 140 kDa with a mean value of 47 kDa (Fig. 2A). - Fig 2 Due to their binding to the negatively charged rRNA, most ribosomal proteins are basic (Fig. 2B). In terms of their isoelectric point (pI), ribosomal proteins from mitochondria are similar to those from ribosomes of the yeast or bacterial cytosol. Except for a few outliers, their pI ranges between 10 and 12. This is considerably higher than the average pI of non-ribosomal matrix proteins where the mean is 8.5. The high pI of MRPs correlates with a high content of positively charged amino acid residues. Since the translocation of positive charges to the negatively charged side of the inner membrane is energetically favored the high content of positive charges might promote matrix translocation of MRPs.

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Eukaryotic cells contain considerably fewer mitochondrial ribosomes than cytosolic ones, particularly upon growth on fermentable carbon sources such as glucose. Hence, MRPs show generally a low abundance, much lower than the average of matrix proteins (Fig. 2C). In summary, ribosomal proteins are relatively homogeneous in respect to their size and positive net charge (Fig. 3). This is also true for MRPs whereas other matrix proteins show significantly more diverse mass and pI values. - Fig 3 -

Presequences of MRPs do often not show the characteristic features of MTSs Most matrix proteins contain N-terminal MTSs that direct them from the cytosol into mitochondria. The probability of an N-terminal region in a given protein to serve as MTS can be predicted with algorithms such as MitoProt II or TargetP (Claros 1995; Emanuelsson et al. 2007; Guda et al. 2004; Reichert and Neupert 2004). MitoProt II predicts probabilities for the presence of an MTS on the basis of a number of different parameters such as number of acidic and basic residues in the N-terminus of a protein, hydrophobicity, presence of putative processing sites and net charge of the entire protein. TargetP is different as it utilizes a neural network to classify proteins into subcellular localizations including mitochondria; the neural network was initially trained on data of Swiss-Prot and the experimental localization annotation of database entries. Most matrix proteins show high probability scores with these prediction programs (median values: MitoProt II 0.985, TargetP 0.867). 80% of all matrix proteins have prediction scores of larger than 0.68 with both algorithms (Fig. 4A). Interestingly, many MRPs show much lower probabilities, particularly with the TargetP program (median values: MitoProt II 0.919, 10


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TargetP 0.624). Only 42% of MRPs show scores of larger than 0.68 with both prediction programs (Fig. 4B). A number of MRPs is not predicted as being matrix proteins by both of these programs (for example Mrp17, Mrpl38, Mrps5, Mrps17, Rsm27, Sws2). This suggests that the mitochondrial targeting information in ribosomal proteins might differ to some degree from that in other matrix proteins. - Fig 4 Unexpectedly, both prediction programs give high probabilities for many cytosolic ribosomal proteins. For example, a third of all proteins of the yeast cytosolic ribosome show MitoProt II values of larger than 0.7. This is even more than for bacterial ribosomes (Fig. 4D) indicating that there was no obvious evolutionary trend to counterselect against MTS-like signals in cytosolic ribosomal proteins (Fig. 4C,D). Some cytosolic ribosomal proteins (for example Rpl2A, Rpl2B, Rpl17A, Rpl17B, Rps13) even yield good scores with both prediction programs. Particularly when considering the high expression levels of these proteins, one might wonder how the mistargeting of these proteins to mitochondria is avoided. Most likely, additional features determine the subcellular localization in ribosomal proteins that still remain to be identified.

Experimental studies on the import of MRPs into mitochondria In vitro import experiments with radiolabeled precursor proteins that were incubated with isolated yeast mitochondria had proved to be very valuable to delineate the mechanisms of mitochondrial protein targeting reactions. Although many matrix proteins were characterized in these assays, precursors of only very few MRPs were tested so far. Most of the few examples that were analyzed, such as Mrpl3 or Mrpl36, were not studied in mechanistic detail

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(Prestele et al. 2009). The only MRPs whose biogenesis was characterized more carefully are Mrpl32 and Mrp10, both showing significant deviations from the classical import route. - Fig 5 -

Unconventional processing of Mrpl32 Mrpl32 is a conserved MRP that is homologous to the E. coli L32 protein. While its MitoProt II score suggests mitochondrial targeting (0.876), it yields only a very low score with the TargetP prediction (0.120). Moreover, despite its length of 71 amino acid residues, the presequence of Mrpl32 is not sufficient to direct proteins into mitochondria and regions of the mature part of Mrpl32 obviously contain crucial targeting information (Nolden et al. 2005). Mrpl32 is imported into the mitochondria in a membrane potential-dependent manner where the N-terminal 71 amino acid residues are proteolytically removed (Grohmann et al. 1991; Nolden et al. 2005). Peculiarly, Mrpl32 is not processed by MPP but rather by the membraneassociated m-AAA protease (Fig. 5A). This hexameric protease is homologous to the bacterial FtsH protease and plays an important role in the degradation of misfolded proteins and in mitochondrial quality control (Arlt et al. 1998; Leonhard et al. 1996; Tatsuta and Langer 2008). In addition to its role in protein degradation it serves as a processing enzyme for some inner membrane proteins (Esser et al. 2002; Koppen et al. 2009; Tatsuta et al. 2007). Mrpl32 is the only known soluble example of m-AAA-mediated processing. Why Mrpl32 uses such an exceptional processing mechanism is not entirely clear, however, the presequence of Mrpl32 is critical for its folding so that the processing by MPP during the import reaction has to be avoided (Nolden et al. 2005). Folding of Mrpl32 relies on four conserved cysteine residues in its structure which presumably bind to zinc or another metal ion. The m-AAA 12


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protease matures Mrpl32 only after its complete import and folding (Bonn et al. 2011); if efficient folding of newly imported Mrpl32 is prevented, such as under oxidative stress conditions, the m-AAA protease degrades the protein rapidly instead of processing it. Hence, the folding rate of Mrpl32 obviously determines whether the m-AAA protease serves as a processing or a degrading enzyme. The unconventional processing of Mrpl32 might therefore be used to control mitochondrial protein synthesis and to regulate the amounts of mitochondrial ribosomes. This control mechanism presumably is conserved from yeast to man and mutations in the m-AAA protease were shown to cause mitochondrial protein synthesis defects in patients suffering from spinocerebellar ataxia (Almajan et al. 2012; Atorino et al. 2003; Nolden et al. 2005).

The unconventional import route of Mrp10 Mrp10 (also called CHCHD1 in mammals) is a small MRP that is found in mitochondria of fungi and animals but has no homologs in bacteria (Koc et al. 2013; Longen et al. 2014; Smits et al. 2007). It uses a highly unconventional import pathway that deviates from that of all other so far characterized matrix proteins (Fig. 5B). The N-terminal region of Mrp10 is very rich in proline residues making it unlikely that it forms an amphipathic helix as it is typically found in MTSs. Accordingly, its TargetP score is very low (0.386), whereas the MitoProt II score (0.840) suggests mitochondrial targeting. Nevertheless, the N-terminal 29 residues obviously serve as MTS since they are sufficient to direct a fused reporter protein into the matrix. Mrp10 contains four highly conserved cysteine residues which are oxidized during its mitochondrial import in the IMS by the Mia40 oxidoreductase (Longen et al. 2014). This oxidoreductase normally drives protein import of IMS proteins thereby introducing disulfide bonds into their structure in a substrate-specific manner (Allen et al. 2005; Chacinska et al. 2004; Mesecke et al. 2005; Naoe et al. 2004; Peleh et al. 2014). Mrp10 is the only matrix 13


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protein known so far that is oxidized by Mia40. It is conceivable that the proline-rich MTS of Mrp10 prevents the tethering of the TOM and TIM23 translocases to allow the binding of Mrp10 to the IMS-located Mia40. This is supported by the observation that when Mrp10 is imported with a “conventional” MTS its cysteine residues bypassed Mia40 and remained reduced (Longen et al. 2014). Despite the presence of disulfide bonds in its structure, Mrp10 is translocated across the inner membrane, presumably through the translocation pore of the TIM23 complex. MPP does not recognize the MTS of Mrp10 as a presequence, so that Mrp10 is not proteolytically processed in the matrix. Following import, Mrp10 assembles into the small subunit of the mitochondrial ribosome where it plays a stabilizing function (Jin et al. 1997). The oxidation of Mrp10 by Mia40 is not essential for its function and a Mrp10 variant in which all four cysteine residues were replaced by serines was still functional. However, the disulfide bonds in Mrp10 make the protein more stable against proteolysis. The reduced form is rapidly degraded in the matrix, particularly at elevated temperatures. Since cytochrome c oxidase is critical to pass electrons on the final electron acceptor of the mitochondrial disulfide relay, molecular oxygen, mutants in this enzyme of the respiratory chain show reduced levels of Mrp10 (Longen et al. 2014). It is conceivable that the unconventional import pathway of Mrp10 is used as a control mechanism to ensure that efficient ribosome assembly only occurs in mitochondria that produce functional cytochrome c oxidase, which contains three of the few translation products of mitochondrial ribosomes. Although at first glance the import processes of Mrpl32 and Mrp10 differ in many of their features, both cases show some striking parallels: folding of both proteins depends on conserved cysteine residues and is crucial to prevent rapid degradation of the proteins. Oxidative conditions that interfere with cysteine-dependent folding prevent the accumulation of Mrpl32 and Mrp10 in mitochondria. It is conceivable that their specific import processes 14


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are used to prevent ribosome assembly under conditions of increased oxidative stress on either site of the inner membrane.

Outlook Our current knowledge on mitochondrial protein import is largely deduced from studies on a number of matrix proteins, however, the biogenesis of MRPs was hardly analyzed. The recent detailed characterization of the import processes of two MRPs, Mrpl32 and Mrp10, showed surprising deviations from the general import pathway. Interestingly, presequences of many MRPs do not show the typical features of MTSs. This might indicate that the import of matrix proteins, in particular of those that are parts of the mitochondrial ribosome, is much more hetergeneous than previously thought. It will be very important in the future to analyze the processes by which MRPs are imported into the matrix and assembled to form mitochondrial ribosomes more systematically.

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Figure Captions Figure 1: Protein import into the mitochondrial matrix. The import reaction can be separated into individual steps: 1, Association of preproteins with cytosolic chaperones. 2, Binding of preproteins to the receptors of the TOM complex. 3, Preprotein translocation through the TOM pore. 4, Membrane potential and ATP-dependent translocation through the TIM23 complex. 5, MPP-mediated processing of preproteins in the matrix. 6, Folding and assembly in the matrix, here shown for a ribosomal protein.

Figure 2: Most MRPs are of small mass, positively charged and of low abundance. A, B. Mass and isoelectric point (pI) distribution of the indicated protein groups. Since maturation sites are in several cases not known with high confidence or are ambiguous, values were calculated from precursor forms including the presequences. For non-ribosomal proteins, matrix proteins were extracted from the list of N-terminally processed mitochondrial proteins published by Vögtle et al. (Vögtle et al. 2009); membrane proteins of unknown topology or with their N-termini facing the IMS were removed. Black dots show mean values. Boxes indicate median values and distances of the 25th and 75th percentiles: whiskers indicate the 5th and 95th percentiles. C. The abundance of proteins on the basis of the numbers given by Ghaemmaghami et al. was plotted (Ghaemmaghami et al. 2003). It should be kept in mind that due to the proteome-wide analyzes there might be considerable error for individual numbers. Moreover, the analysis was done upon growth on glucose where expression of mitochondrial protein is largely repressed. Nevertheless, it is obvious that MRPs are in general relatively inabundant proteins in yeast, whereas cytosolic ribosomal proteins often are present with several 10,000 copies per cell.

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Figure 3: Ribosomal proteins show a relatively narrow mass and pI distribution. A-D. Mass and pI values are shown on scatter plots. To facilitate a better comparison of the point clouds representing the individual datasets in regard to concentration, density contours are additionally shown in the corresponding plots, and are chosen to reflect quartiles of the data, i.e. they encompass 25%, 50%, and 75% of measurements, from outside to inside. The contours are determined as level sets of a Gaussian kernel density estimate obtained using rule-of-thumb bandwidth selection (Venables and Ripley 2002). These plots were generated using the R software for statistical computing. Data points for proteins larger than 80 kDa are not shown (only applicable for panel A).

Figure 4: Prediction programs often fail to detect the MTSs in MRPs. A-D. Sequences of the indicated proteins were used to calculate probability scores for mitochondrial targeting using MitoProt II and TargetP (Emanuelsson et al. 2007; Guda et al. 2004; Reichert and Neupert 2004). Density contours were calculated and are shown as described for Fig. 3. Boxes depict the region in which sequences show scores of larger than 0.68 with both algorithms. The percentages of data points in these areas are indicated.

Figure 5: Import pathways of Mrpl32 and Mrp10. A. Mrpl32 is imported into the matrix where it folds in a process that involves the binding of a metal ion, most likely zinc, to cysteine residues. Subsequently, it is proteolytically processed by the m-AAA protease before it assembles into the ribosome. B. Mrp10 is initially imported into the IMS where it binds to Mia40. Two disulfide bonds are formed in Mrp10 by the mitochondrial disulfide relay system that consists of Mia40, Erv1, cytochrome c and cytochrome c oxidase. In a subsequent

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reaction, Mrp10 traverses the inner membrane into the matrix where it assembles into the

ribosome.

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Table 1. Yeast MRPs of the large subunit

Nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Gene Name MRPL3 RML2 MRP7 MRPL35 MRPL4 MRPL10 MRPL22 MRPL40 MRPL7 MRPL17 YML6 MRPL1 MRP20 MRPL13 MRPL24 MRPL9 MRPL11 MRPL15 MRPL8 MRPL16 MRPL6 MRPL20 MRPL32 MNP1 MRPL36 IMG1 MRPL25 MRPL23 MRPL49 MRPL28 MRPL19 MRPL27 IMG2 MRPL50 MRPL51 MRP49 MRPL31 MRPL38 MRPL37 MRPL44 RTC6 MRPL33

Systematic Name YMR024W YEL050C YNL005C YDR322W YLR439W YNL284C YNL177C YPL173W YDR237W YNL252C YML025C YDR116C YDR405W YKR006C YMR193W YGR220C YDL202W YLR312W-A YJL063C YBL038W YHR147C YKR085C YCR003W YGL068W YBR122C YCR046C YGR076C YOR150W YJL096W YDR462W YNL185C YBR282W YCR071C YNR022C YPR100W YKL167C YKL138C YKL170W YNL122C YDR115W YBR268W YMR225C YPL183W-A YMR286W

E. coli Homolog L2 L27 L29 L15 L22 L24 L5 L4 L1 L23 L28 L3 L10 L17 L16 L6 L32 L7/L12 L31 L19 L13 L21 L11

L9

L14 L35 L34

L36 L30

Mass 43999 43786 43226 42825 36965 36347 34897 33751 33099 32212 31969 30996 30566 30271 30049 29790 28515 28167 26945 26518 23889 22392 21437 20650 20091 19394 18587 18463 18387 17342 16670 16492 16380 16283 16124 16021 15520 14904 13244 12030 11949 11476 10698 9531

pI 10.24 11.50 10.63 10.34 7.69 11.17 10.61 10.26 10.47 9.87 10.45 10.78 10.24 9.61 10.95 10.99 10.51 9.95 10.60 11.12 10.70 11.00 10.65 9.97 10.53 11.19 10.87 10.91 11.53 11.27 10.68 10.96 10.66 9.15 11.20 10.15 11.46 10.57 12.77 12.53 10.67 10.38 11.89 11.00

MitoProt II Score1 0.949 0.992 0.829 0.911 0.642 0.987 0.890 0.889 0.987 0.914 0.656 0.878 0.514 0.984 0.790 0.920 0.529 0.982 0.834 0.884 0.987 0.919 0.876 0.983 0.980 0.989 0.686 0.272 0.993 0.975 0.460 0.944 0.885 0.982 0.919 0.940 0.724 0.265 0.998 1.000 0.980 0.683 0.990 0.915

1, Claros 1995; 2, Emanuelsson et al. 2007; 3, Experimentally verified by Vögtle et al. 2009

TargetP Score2 0.788 0.726 0.516 0.764 0.423 0.564 0.920 0.624 0.796 0.675 0.624 0.371 0.482 0.835 0.853 0.738 0.625 0.886 0.410 0.426 0.748 0.559 0.120 0.871 0.670 0.873 0.394 0.798 0.767 0.493 0.083 0.337 0.596 0.732 0.759 0.609 0.621 0.092 0.416 0.915 0.856 0.361 0.711 0.564

N-Terminal Residue3 ? ? 32 ? ? 58 35 2 ? ? 17 29 35 ? 36 ? 32 29 2 38 17 ? 72 11 16 ? 2 ? 45 59 55 81 39 2 2 ? ? ? ? ? ? ? ?

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Table 2, Yeast MRPs of the small subunit

Nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Gene Name RSM22 NAM9 RSM23 VAR1 PPE1 MRP4 RSM28 MRPS35 MRP51 MRP13 RSM24 MRP1 PET123 MRPS5 MRPS28 MRPS9 RSM25 RSM26 RSM7 MRPS17 MRPS18 RSM10 MRP21 MRPS8 MRPS12 SWS2 RSM18 MRP17 YMR31 MRPS16 MRP2 RSM27 MRP10 RSM19

Systematic E. coli MitoProt II TargetP N-Terminal Name Homolog Mass pI Score1 Score2 Residue3 YKL155C 72190 10.20 0.967 0.577 25 YNL137C S4 56356 10.46 0.984 0.726 54 YGL129C S29 50867 10.32 0.998 0.836 ? Q0140 S3/S24 47123 10.75 0.956 0.118 ? YHR075C 44887 7.05 0.457 0.156 ? YHL004W S2 44151 9.59 0.995 0.941 ? YDR494W 41216 10.77 0.826 0.615 15 YGR165W 39575 10.72 0.969 0.854 ? YPL118W 39445 10.76 0.293 0.507 2 YGR084C 38988 10.26 0.947 0.457 68 YDR175C S35 37393 10.05 0.890 0.741 31 YDR347W 36728 9.81 0.834 0.696 13 YOR158W 35998 10.61 0.990 0.507 2 YBR251W S5 34883 10.41 0.520 0.447 14 YDR337W S15 33057 10.72 0.989 0.804 34 YBR146W S9 31925 11.07 0.993 0.943 46 YIL093C S23 30513 6.21 0.418 0.322 1 YJR101W 30223 9.47 0.982 0.595 74 YJR113C S7 27816 10.54 0.959 0.923 ? YMR188C S17 27635 10.38 0.504 0.475 42 YNL306W S11 24563 10.71 0.987 0.887 ? YDR041W S10 23424 10.10 0.848 0.716 ? YBL090W S21 20394 11.28 0.987 0.811 25 YMR158W S8 17470 10.37 0.950 0.383 ? YNR036C S12 16917 11.80 0.999 0.903 ? YNL081C S13 16089 11.21 0.350 0.137 ? YER050C S18 15835 11.53 0.761 0.367 ? YKL003C S6 15021 10.66 0.116 0.116 72 YFR049W S36 13689 10.30 0.744 0.369 9 YPL013C S16 13639 11.18 0.968 0.874 2 YPR166C S14 13538 11.70 0.568 0.785 17 YGR215W S33 12393 11.02 0.037 0.247 1 YDL045W-A 10690 10.70 0.840 0.386 ? YNR037C S19 10275 11.32 0.924 0.726 25

1, Claros 1995; 2, Emanuelsson et al. 2007; 3, experimentally verified by Vögtle et al. 2009


Page 26 of 30

cytosolic Hsp70

ATP

matrix Hsp70 ADP

1

ADP ADP

3 2

5

Cytosol OM

TOM IMS

TIM23 4 IM

and other matrix proteases

MPP Matrix

ADP

6

?

Ribosome Assembly

A

C

Non-ribosomal proteins

Ribosomal proteins

140 120

Mass [kDa]

100

Ribosomal proteins

80 60 40 20 0

B

Matrix Yeast

Non-ribosomal proteins 14

Matrix Yeast

Cytosol Yeast

Non-ribosomal proteins

Cytosol E. coli 80000

Ribosomal proteins 60000

Abundance [molecules / cell]

12 10

pI


Page 27 of 30

8

40000

20000

6 4

Matrix Yeast

Matrix Yeast

Cytosol Yeast

Cytosol E. coli

0

Matrix Yeast

Matrix Yeast

Cytosol Yeast

pI

pI

pI

pI


Page 28 of 30

A

0

C

0

Matrix proteins without MRPs

20

20 40 Mass [kDa]

40 Mass [kDa] 60

60

B

10

5

0 80

5

0 80

0

Cytosolic ribosomal proteins

D

10

0

MRPs

10

5

0 20

20

40 Mass [kDa]

40 Mass [kDa]

60

60

80

E. coli ribosomal proteins

10

5

0 80

A

B

Matrix Matrixproteins proteinswithout withoutMRPs MRPs 80% MitoProtpIII Score

0.75 10 0.50 5 0.25

0.00 0 00.00

C

1.00

20 0.25

40 0.50 TargetP Score Mass (kDa)

0.75 60

0.75 10 0.50 5 0.25

0.00 0 00.00

20 0.25

40 0.50 TargetP Score Mass (kDa)

0.75 60

0.50 5 0.25

1.00

4%

1.00 80

42%

0.75 10

D

Cytosolic Cytosolicribosomal ribosomalproteins proteins

MRPs MRPs

1.00

0.00 0 00.00

1.00 80

MitoProtpIII Score

MitoProtpIII Score

1.00

MitoProtpIII Score


Page 29 of 30

20 0.25

0.50 40 TargetP Score Mass (kDa)

0.75 60

1.00 80

E. E.coli coliribosomal ribosomalproteins proteins 9%

0.75 10 0.50 5 0.25

0.00 0 00.00

20 0.25

0.50 40 TargetP Score Mass (kDa)

0.75 60

1.00 80

A

Mrp10

B

Mrpl32

Translocation across the outer membrane

Import

Cytosol OM

Oxidation-dependent folding

SS

SS SS

SS

m-AAA Protease

Mia40

Erv1 Cytochrome c

IMS

TIM23

ATP

IM Processing

Cytochrome c oxidase

Matrix ATP

SS SS

Zn ADP ADP

Redox-sensitive folding

Zn ADP ADP

SS SS


Page 30 of 30

Ribosome Assembly

Ribosome Assembly

SS SS

Translocation across the inner membrane

Import of proteins into mitochondria.

Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria.

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import.

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis.

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

The nuclear import of ribosomal proteins is regulated by mTOR.

Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria.

Role of membrane contact sites in protein import into mitochondria.

Identification of a receptor for protein import into mitochondria.

Phospholipid import into mitochondria: possible regulation mediated through lipid polymorphism.

In vitro synthesis of yeast ribosomal proteins.

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

Mitochondria-targeted RNA import.

The Import of Proteins into the Mitochondrion of Toxoplasma gondii.

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms.

Transport of proteins into mitochondria and chloroplasts.

Import of proteins into peroxisomes and other microbodies.

Nuclear Import of Yeast Proteasomes.

Cellular site of synthesis of ribosomal proteins in yeast.

Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl.

Structural and functional analysis of yeast ribosomal proteins.

Yeast ribosomal proteins are synthesized on small polysomes.