Fungal Genetics and Biology 36 (2002) 85–90 www.academicpress.com
Review
Protein import into mitochondria of Neurospora crassa Holger Prokisch,* Stephan Nussberger, and Benedikt Westermann Institut f€ur Physiologische Chemie, Universit€ at M€unchen, Butenandtstr. 5, 81377 Munich, Germany Received 8 March 2002; accepted 14 March 2002
Abstract Biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mitochondrial structure and function. Import and sorting of these preproteins is a multistep process facilitated by complex proteinaceous machineries located in the mitochondrial outer and inner membranes. The translocase of the mitochondrial outer membrane, the TOM complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel formed by TOM40. The TOM complex is able to insert resident proteins into the outer membrane and to translocate proteins into the intermembrane space. For import of inner membrane or matrix proteins, the TOM complex cooperates with translocases of the inner membrane, the TIM complexes. During the past 30 years, intense research on fungi enabled the identification and mechanistic characterization of a number of different proteins involved in protein translocation. This review focuses on the contributions of the filamentous fungus Neurospora crassa to our current understanding of mitochondrial protein import, with special emphasis on the structure and function of the TOM complex. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Biogenesis; Mitochondria; Neurospora crassa; Protein import; TOM; TIM
1. Introduction Mitochondria supply the cell with energy generated by oxidative phosphorylation (Saraste, 1999). Furthermore, they are the compartment for many other important metabolic processes, including reactions of the tricarboxylic acid cycle, iron/sulfur cluster assembly, and biosynthesis of many cellular metabolites (Scheffler, 2001). Mitochondria cannot be generated de novo, but grow from preexisting organelles. Despite the capacity of mitochondria to encode and synthesize polypeptides, the major part of their proteome is encoded by a vast array of genes located in the nucleus. In fungi, as in most eukaryotic organisms, only a handful of genes were retained in the mitochondrial genome during evolution (Gray et al., 1999). These encode mostly hydrophobic components of the respiratory chain and ribosomal subunits. Several hundred mitochondrial proteins are synthesized on cytosolic ribosomes and have to be imported into mitochondria. This requires complex protein
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[email protected] (H. Prokisch).
machineries for the import and sorting of polypeptides (Neupert, 1997). The molecular machinery that mediates protein translocation into mitochondria has been conserved during the evolution of eukaryotic cells. Fungi are excellent model organisms for studying this process because genetic and biochemical approaches can be readily combined. Distinct advantages of the filamentous fungus Neurospora crassa and the budding yeast Saccharomyces cerevisiae enabled major discoveries of mitochondrial biology. The characterization of the mitochondrial protein import complexes in yeast has been discussed in several excellent recent reviews (Herrmann and Neupert, 2000; Neupert, 1997; Pfanner and Geissler, 2001). Why has Neurospora proven to be an extremely valuable model organism for the study of mitochondrial biogenesis? Early genetic studies certified Neurospora as a genetically tractable organism and demonstrated that its haploid constitution, easy culture conditions, and susceptibility to mutagenesis make it an ideal eukaryotic system for genetic studies that allow the accumulation of a wealth of biological and genetic information (Davis, 2000; Perkins, 1992; Perkins and Davis, 2000). In the past 3 decades especially, the
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possibility of obtaining large amounts of Neurospora organelles in a straightforward and economic way allowed the identification and detailed biochemical characterization of a number of mitochondrial proteins. Here, we review the contributions of Neurospora to our current understanding of the cellular mechanisms of protein import into mitochondria.
2. Mitochondrial protein import pathways and receptors Import of cytosolically synthesized mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM1 complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. The TOM complex must specifically recognize mitochondrial precursor proteins synthesized in the cytosol and translocate them across the outer membrane (Fig. 1). Moreover, it has to insert resident proteins into the outer membrane. For further import into or across the inner membrane the TOM complex cooperates with the TIM complexes. The TIM23 complex mediates import of preproteins with usually N-terminal positively charged targeting signals, whereas the TIM22 complex in cooperation with soluble complexes of small TIM proteins in the intermembrane space (tiny TIMs) facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane (Fig. 1). Almost 25 years ago, kinetic studies on import of the intermembrane space protein cytochrome c using intact cells of N. crassa showed that newly synthesized mitochondrial precursor proteins first exist in an extramitochondrial pool from which they are posttranslationally imported into mitochondria (Hallermayer et al., 1977). Employing an in vitro import assay it was demonstrated in a cell-free system that the translocation of precursor proteins into mitochondria is not mechanistically coupled to their synthesis on cytosolic polysomes (Harmey et al., 1977), that different receptors on the mitochondrial surface mediate the specific recognition of precursor proteins (Zimmermann et al., 1981), and that a membrane potential is required for the posttranslational transfer of proteins into mitochondria (Schleyer et al., 1982). Later, the study of the import of three different precursor proteins, apocytochrome c, ADP/ATP carrier (AAC), and subunit 9 of the F0 -ATPase (Su9), revealed the existence of distinct import pathways. Apocytochrome c is imported by the TOM complex directly into 1 Abbreviations used: TOM, translocase in the outer membrane; TIM, translocase in the inner membrane; AAC, ADP/ATP carrier; Su9, subunit 9 of the F0 -ATPase; CCHL, cytochrome c heme lyase; RIP, repeat-induced point mutation.
Fig. 1. Schematic model of the mitochondrial protein import machinery and different pathways of protein translocation into mitochondria. Preproteins carrying a positively charged presequence at their amino terminus (left) are targeted to the Tom20 receptor, passed over Tom22 to the protein conducting channel formed by Tom40, and translocated across the outer membrane. In cooperation with the TIM23 translocase these preproteins are further translocated into or across the inner membrane. The Tom70 receptor recognizes internal targeting signals of preproteins, such as the members of the mitochondrial carrier family (right). After their transfer and insertion into the TOM pore, a soluble complex of tiny TIMs guides them to the TIM22 complex, which mediates the insertion of the preprotein into the inner membrane. (+++), positively charged presequence; OM, outer membrane; IMS, intermembrane space; IM, inner membrane; Dw, electrochemical potential across the inner membrane.
the intermembrane space (Diekert et al., 2001). Its import requires the conversion to the holoenzyme by the intermembrane space protein cytochrome c heme lyase, CCHL (Drygas et al., 1989; Nargang et al., 1988; Nicholson et al., 1988). The precursor of Su9 contains a characteristic N-terminal presequence, which forms an amphipathic, positively charged helix and is processed by the matrix processing peptidase, MPP (Arretz et al., 1994). Import of Su9 is dependent on an electrochemical potential across the mitochondrial inner membrane (Schmidt et al., 1983). In contrast, AAC contains internal targeting signals and is not processed. Insertion into the inner membrane, however, also depends on a membrane potential across the inner membrane (Zwizinski et al., 1983). Recent work with yeast suggests that these different import characteristics reflect the use of different translocation machineries, namely, the TIM23 complex for proteins carrying a cleavable N-terminal presequence, such as Su9, and the TIM22 complex for inner membrane proteins with internal targeting signals, such as AAC (Herrmann and Neupert, 2000). Subsequently, N. crassa has helped to identify and unravel the structure and function of a number of components involved in targeting of nuclear-encoded preproteins to mitochondria. This was facilitated by the isolation of mitochondrial outer membrane vesicles and
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the generation of antibodies to outer membrane components. Using these antibodies the two major import receptors, Tom20 and Tom70, were identified and their substrate specificities characterized. While antibodies against Tom20 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, but not AAC (S€ ollner et al., 1989), antibodies against Tom70 selectively repress import of AAC at the level of specific binding to mitochondria, but do not interfere with the import of precursors with N-terminal targeting signals (S€ ollner et al., 1990). Furthermore, Tom70-deficient mitochondria revealed a specific defect in the uptake of carrier proteins with internal targeting signals (Grad et al., 1999). The investigation of the interactions of the two import receptors with other outer membrane proteins led to the identification of four further proteins as components of the TOM complex, namely, Tom6, Tom7, Tom22, and Tom40 (Kiebler et al., 1990; S€ ollner et al., 1992).
3. Molecular mechanism of protein transport across the mitochondrial outer membrane The TOM and TIM machineries act in concert during the translocation of matrix and inner membrane proteins. In intact mitochondria, the membrane potential across the inner membrane is strictly required for import of preproteins with N-terminal presequences, even at an early translocation step across the outer membrane. However, the TOM and TIM complexes act as distinct entities in a sequential reaction. Using a fusion protein with bivalent targeting information it was demonstrated that in the absence of a membrane potential the protein translocates into the intermembrane space, but upon reestablishment of the membrane potential it can be further chased into the matrix (Segui-Real et al., 1993). Therefore it was reasonable to use highly purified outer membrane vesicles for mechanistic studies of protein transport into or across the mitochondrial outer membrane. This experimental system allowed the insertion of outer membrane proteins into the membrane and translocation of an intermembrane space protein, CCHL, into the lumen of the vesicle (Mayer et al., 1993). In contrast, preproteins carrying N-terminal presequences were not fully imported into outer membrane vesicles. It was observed, however, that the presequence portion becomes exposed to the intermembrane space. The initial interaction of a presequence with the mitochondrial import machinery occurs by binding to the surface-exposed cis site (Mayer et al., 1995b; Rapaport et al., 1998b). Preproteins bound to the cis site can be cross-linked via their N-terminal presequence to Tom20, Tom22, and Tom40, demonstrating a direct association of this part of the preprotein with the import machinery
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(Rapaport et al., 1997). Tom22 functions as a second receptor, mediating the transfer of preproteins from Tom20 to the translocation pore (Kiebler et al., 1993; Mayer et al., 1995a). The interaction with the cis site is only transient and is immediately followed by the insertion of the polypeptide chain into the translocation pore. The presequence then stably associates with a second specific binding site located at the inner face of the outer membrane, termed the trans site (Mayer et al., 1995b; Rapaport et al., 1998b). The insertion and translocation of the precursor are concomitant with distinct structural alterations of the TOM complex (Rapaport et al., 1998a). Binding of precursor proteins to the trans site is mediated mainly by the presequence and is accompanied by unfolding of the protein on the cytosolic side of the membrane (Court et al., 1996; Mayer et al., 1995b; Nargang et al., 1998; Rapaport et al., 1997; Stan et al., 2000). Binding to the trans site is strong enough to shift the equilibrium to the side of translocation and is a prerequisite for further recognition and transport of the precursor protein across the inner membrane, which is then facilitated by the TIM23 translocase (Mayer et al., 1995b; Rapaport et al., 1998b; Stan et al., 2000). N. crassa has proven to be an excellent organism for biochemical approaches to study protein import into mitochondria. This was contrasted by the relative difficulty of isolating mutants in specific target genes, especially for genes that are essential for cell viability. Novel genetic methods had to be developed to analyze the function of the essential TOM components (Metzenberg and Grotelueschen, 1992). These techniques turned out to be generally useful for the isolation of mutants in genes that code for essential functions. The ‘‘sheltered RIP’’ technique was used to create a knockout mutant for tom20. This approach makes use of repeat-induced point mutations, RIP (Selker, 1990), for destruction of the target gene, but allows the maintenance of mutated alleles in a heterokaryon in which the normal copy of the gene, present in another nucleus, shelters the cell against potentially lethal effects of the mutation (Harkness et al., 1994). In an alternative approach, the newly developed procedure of ‘‘sheltered disruption’’ was employed to obtain a knockout strain for tom22. Here, the gene was disrupted via homologous recombination in a heterokaryotic strain. The mutant strain harbors two nuclei, one with a null allele of the tom22 gene and another with a wild-type allele (Nargang et al., 1995). In both techniques, the nucleus harboring the destroyed gene contains a selectable marker that enables the shift of nuclear ratios in the heterokaryons. Selection for nuclei containing the knockout alleles results in strongly reduced levels of the target gene product and thus allows the study of the mutant phenotype. Deficiency in Tom20 or Tom22 resulted in a severe growth defect, indicating
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that both proteins are essential and fulfill important roles in the biogenesis of mitochondria. This is in contrast to the situation in yeast where both deletion mutants are viable (Ramage et al., 1993; van Wilpe et al., 1999). The investigation of mutants deficient in TOM receptor proteins confirmed that Tom70 and Tom20 have distinct precursor specificities and that Tom22 is essential for translocation of the majority of preproteins (Harkness et al., 1994; Nargang et al., 1995; Grad et al., 1999). The purification of outer membrane vesicles from N. crassa on a large scale followed by affinity purification of the TOM holo complex from a strain that carried a version of Tom22 with a hexahistidinyl tag at its C terminus enabled a detailed biochemical and biophysical characterization in its native form (K€ unkele et al., 1998a,b). The TOM holo complex contains seven subunits: Tom70, Tom40, Tom22, Tom20, Tom7, Tom6, and Tom5 (Dembowski et al., 2001; K€ unkele et al., 1998a). Upon reconstitution into liposomes, the TOM complex mediated integration of mitochondrial outer membrane proteins into and translocation of intermembrane space proteins across the membrane. This demonstrates the functional integrity of the purified complex and indicates that the TOM complex is sufficient for protein translocation into or across the outer membrane (Diekert et al., 2001; K€ unkele et al., 1998a).
Fig. 2. Three-dimensional map of the protein translocation machinery of the outer membrane of mitochondria (TOM core complex) obtained by electron tomography. The TOM core complex was purified from mitochondria of N. crassa. Negatively stained TOM core complex particles were reconstructed individually before three-dimensional alignment and averaging was performed. The overall size of the TOM core complex is roughly 13–14 nm. The reconstruction exhibits two channels traversing the complex with a diameter of ca. 2.1 nm and a height of 7 nm. (Image reproduced from Ahting et al. (1999) by copyright permission of The Rockefeller University Press, New York.)
core or the TOM holo complex (Ahting et al., 2001, 1999; K€ unkele et al., 1998a,b). Detailed biophysical characterization demonstrated that Tom40 is the structural key component of the TOM complex and indicates that Tom40 can form the protein translocation channel of the mitochondrial outer membrane. Mechanistic studies will reveal in the future how the structure of the TOM complex relates to its function.
5. Perspectives 4. Structure of the TOM complex The possibility of obtaining large quantities of highly purified TOM complex allowed a first view of its structure. The filtered electron microscopic images of negatively stained particles revealed structures about 13 nm in diameter with one, two, or three stain-filled holes 2 nm in diameter. The projection map revealed two or three centers of stain accumulation in most of the particles. Two of these centers might represent channels in the complex through which a translocating polypeptide would be threaded in situ (K€ unkele et al., 1998a). It is conceivable that the third density in the holo complex is due to the presence of the receptors, Tom20 and Tom70, which are easily released in the presence of mild detergents (Ahting et al., 1999). Electron tomography and three-dimensional image reconstitution of the TOM core complex, which lacks these receptor components, yielded a map with two channels traversing the complex (Fig. 2). Further removal of the remaining TOM components from Tom40 by a different detergent results in a high-molecular-mass complex formed by Tom40 alone (Ahting et al., 2001). Electrophysiological single-channel measurements confirmed that channels do exist in all complex preparations and that the recorded Tom40 channels are very similar to those found with the TOM
In the past 30 years, N. crassa emerged as a powerful experimental system for studying different aspects of mitochondrial biology (see accompanying review for a discussion of mitochondrial dynamics). For the investigation of protein import into mitochondria, Neurospora helped to set several milestones, such as the characterization of mitochondrial protein import in vitro, the identification of TOM components, and most recently the functional reconstitution and structural analysis of the TOM complex. Genomic sequence analysis helped in the identification of components of the TIM complexes. The next challenge is the biochemical and biophysical characterization of the protein sorting machineries of the inner membrane isolated in their native state.
Acknowledgments We thank the members of our laboratories for their invaluable work, Walter Neupert for continuous support, and Johannes Herrmann and Uwe Ahting for critical comments on the manuscript. The authors’ work is supported by the Deutsche Forschungsgemeinschaft through grants SFB 549/B4 (to H.P.), and SFB 413/B3 and WE 2174/2-1 (to B.W.) and by the
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Bundesministerium f€ ur Bildung und Forschung through Grant MITOP (to H.P.).
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