The mitochondrial processing peptidase

ht. J. Biochem.

Pergamon PII: 31357~2725@7)00032-0

Cell Bid. Vol. 29, No. S/9, pp. 1043-1045,

1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 1357-2725/97 $17.00 + 0.00

MOLECULES IN FOCUS

The Mitochondrial Processing Peptidase HANS-PETER BRAUN, UDO KLAUS SCHMITZ” Institut fiir Angewandte Genetik, Universitiit Hannover, Herrenhiiuserstr. Germany

2, D-30419, Hannover,

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which plays an essential role in mitochondrial protein import. It cleaves off the N-terminal targeting signals of nuclear encoded mitochondrial proteins upon their transport into theorganege. In mammals and yeast the enzyme is localixed in the mitochondrial matrix while in plants it is integrated into a protein complex of tbe respiratory chain. As the activity of MPP is essential for the viability of eukaryotic cells it is conceivable that inhibitors of MPP which are specific for tbe soluble enzyme only present in fungi and animals may work as fungicides or insecticides.0 1997 Elsevier Science Ltd Keywords: Protein processing Protein import bc, complex Mitochondria Int. J. Biochem. Cell Biol. (1997) 29, 1043-1045

INTRODUCTION

MPP (EC 3.4.24.64) was the first component of the mitochondrial protein import apparatus to be identified (Biihni et al., 1980) but its successful purification was not reported before 1988 (Hawlitschek et al., 1988; Yang et al., 1988). The enzyme catalyses the proteolytic cleavage of N-terminal signals, termed presequences, which target nuclear encoded proteins into mitochondria (Fig. 1). As MPP from fungi and mammals is supposed to form a soluble heterodimer in the mitochondrial matrix it was initially termed matrix processing peptidase. In 1992 it turned out that MPP from plants is localized not in the matrix space but in a protein complex of the inner mitochondrial membrane (Braun et al., 1992). Surprisingly, in potato, wheat and other plants the two non-identical subunits of MPP substitute the so-called ‘core’ proteins of cytochrome c reductase (also termed bc, complex), a protein complex of the respiratory chain. The name ‘core’ proteins alludes to the initial assumption that these high molecular weight subunits form the center of the bc, complex. Today it is clear that they are *To whom all correspondence should be addressed. Received 9 July 1996; accepted 10 March 1997.

peripherally localized and not involved in electron transfer. The integration of MPP in a protein complex of the inner mitochondrial membrane led to a new naming of the enzyme as ‘mitochondrial processing peptidase’ instead of ‘matrix processing peptidase’ (Emmermann et al., 1993). In the organisms analysed different names were given to the two subunits of MPP (the smaller subunit is always given first): PEP (processing enhancing protein) and MPP in Neurospora (Hawlitschek et al., 1988), Masl and Mas2 in yeast (Yang et al., 1988) and P-52 and P-55 in rat (Ou et al., 1989). According to the new and generally accepted nomenclature, the components of the enzyme are designated a-‘MPP and j?-MPP (Kalousek et al., 1993). From the early eighties it was assumed that MPP is a metal-dependent endopeptidase but Ou et al. (1989) were the first to show that the presequences of mitochondrial precursors are indeed removed in a single cleavage step. STRUCTURE

The two subunits forming the MPP holoenzyme are structurally related. The larger subunit, cr-MPP, has a molecular weight of 53-57 kDa while the size of /I-MPP ranges

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Hans-Peter Braun and Udo Klaus Schmitz

between 48 and 52 kDa. Only in plants the j?-MPP subunit (53-55 kDa) is larger than CC-MPP (51 kDa). Together with the ‘core’ proteins of cytochrome c reductase both MPP subunits belong to the pitrilysin family of metalloendopeptidases. The members of this family are characterized by an inverse zinc-binding site and play important roles in cellular metabolism and the biogenesis of mitochondria and chloroplasts. Interestingly a-MPP and the ‘core’ proteins have incomplete zinc-binding sites while b-MPP contains an intact inverse zinc binding domain and was shown to be the catalytically active subunit (Braun and Schmitz, 1995; Kitada et al., 1995). In potato, both subunits of MPP are present in two isoforms, termed c( l-MPP, a2-MPP, fl I-MPP and /?2MPP (Emmermann et al., 1993). SYNTHESIS AND DEGRADATION

Both MPP subunits are encoded in the nucleus, synthesized on cytoplasmic ribosomes and transported posttranslationally into the mitochondria. The N-terminal targeting signals of a-MPP and fi-MPP are 13-32 amino acids long and have the characteristic features of mitochondrial presequences: they are rich in basic and hydroxylated amino acids and have the potential to form amphiphilic a-helices. The presequences of the protease precursors are removed by the MPP holoenzyme during their import into mitochondria. BIOLOGICAL

FUNCTION

MPP proteolytically removes N-terminal presequences, which are heterogenous in size and primary structure. Their length ranges from

N P-m

/ K

a - m & c

Fig. 1. The heterodimeric mitochondrial processing peptidase (MPP) is just cleaving off the N-terminal portion of a mitochondrial precursor protein (shown in red). The arrow indicates the cleavage site. The letters ‘N’ and ‘C’ denote the amino-terminus and the carboxy-terminus of the precursor protein, respectively.

8 to 69 amino acids and apart from a conserved arginine residue at the P2 or P3 position they lack significant sequence similarity. Mutational analysis of presequences suggests that not the amphiphilic a-helices but rather other parts of presequences are recognized by MPP. The activity of MPP is strictly dependent on the presence of both subunits, a-MPP and B-MPP. In yeast it was shown that the genes encoding them belong to the small group of nuclear sequences that are essential for cell viability. Apart from its function in the processing of mitochondrial presequences MPP may also be involved in the maturation of polyproteins like N-acetylglutamate kinase and N-acetyl-y-glutamyl-phospate reductase. These enzymes are encoded by a single nuclear gene in many fungi, synthesized as a polyprotein and separated by MPP. The processing activity is inhibited by chelators of divalent cations like EDTA and 1, IO-phenantroline but otherwise the activity of MPP from most organisms is insensitive toward inhibitors of cysteine-, serine- or aspartateproteases. Although being integrated into the cytochrome c reductase complex in plants, MPP activity is not affected by inhibitors of electron transfer like antimycin A or myxothiazol. This means that electron transfer and removal of presequences are independent functions of the cytochrome c reductase/processing peptidase complex although in potato both activities cannot be separated by treatment with salt or detergent (Emmermann et al., 1993). It seems likely that the different submitochondrial localization of both activities in autotrophic and heterotrophic organisms reflects different stages of the co-evolution of both enzymes (Fig. 2). Potato and other plants exemplify the original situation in which an ancient protease was associated with the simple prokaryotic bc, complex. Interestingly, Neurospora represents an intermediate stage as the P-MPP substitutes the ‘core I’ protein of the bc, complex and simultaneously is a soluble protein of the matrix that removes presequences together with a-MPP (Schulte et al., 1989). The situation in yeast and beef with a completely soluble heterodimeric MPP in the matrix space probably resulted from a phylogenetically more recent event, which allowed independent regulation of mitochondrial biogenesis and respiration. This also explains why the ‘core’ proteins from yeast and mammals only contain incomplete zinc-binding motifs: they have lost their proteolytic function after it was taken over by the soluble processing

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cell; so in general MPP may be used as an activator of non-functional fusion proteins containing a presequence or an appropriate cleavage site. As MPP is integrated into the cytochrome c reductase complex only in plants, it is conceivable that inhibitors of MPP which are specific for the soluble form only present in fungi and animals may work as fungicides or insecticides. Acknowledgement-This work is supported Forschungsgemeinschaft.

by.the Deutsche

REFERENCES

Neurospora

Yeast, Beef Fig. 2. Different stages in the co-evolution of MPP and the cytochrome c reductase complex (bci complex). (top) Plant mitochondria, where both subunits of MPP are integrated into the respiratory bci complex. (Middle) Neurospora mitochondria, which represent an intermediate stage as the proteolytically active /I-MPP subunit forms still part of the complex but a soluble heterodimeric MPP is already present in the matrix. (Bottom) There is no longer a spatial or functional relationship between the cytochrome c reductase complex and the mitochondrial processing peptidase in yeast and mammals. IMS: mitochondrial intermembrane space, IM: inner mitochondrial membrane, M: mitochondrial matrix.

peptidase. Taken together, the present knowledge on MPP and the bc, complex suggests that the ‘core’ proteins of this respiratory complex are relics of the subunits of MPP (Braun and Schmitz, 1995). INDUSTRIAL

APPLICATIONS

Specific endopeptidases like MPP are essential for activation and transport processes in the

BShni P., Gasser S., Leaver C. and Schatz G. (1980) A matrix-localized mitochondrial protease processing cytoplasmically made precursors to mitochondrial proteins. In: T h e o r g a n i s a t i o n a n d e x p r e s s i o n of t h e m i t o c h o n d r i a l genome, eds A. M. Kroon and C. Saccone, pp. 423433. Elsevier Science Publishers, Amsterdam. Braun H. P. and Schmitz U. K. (1995) Are the ‘core’ proteins of the mitochondrial bc, complex evolutionary relics of a processing protease? Trends in Biochemical Sciences 20, 171-175. Braun H. P., Emmermann M., Kruft V. and Schmitz U. K. (1992) The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain. EMBO Journal 11, 3219-3227. Emmermann M., Braun H. P., Arretz M. and S&m& U. K. (1993) Characterization of the bifunctional cytochrome c reductase/processing peptidase complex from potato mitochondria. Journal of Biological Chemistry 268, 1893618942. Hawlitschek G., Schneider H., Schmidt B., Tropschug M., Hart1 F.-U. and Neupert W. (1988) Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell 53,795806. Kalousek F., Neupert W., Omura T., Schatz G. and Schmitz U. K. (1993) Uniform nomenclature for the mitochondrial proteases cleaving precursors of mitochondrial proteins. Trends in Biochemical Sciences 18, 249. Kitada S., Shimokata K., Niidome T., Ogishima T. and Ito A. (1995) A putative metal-binding site in the B-subunit of rat mitochondrial processing peptidase is essential for its catalytic activity. Journal of Biochemistry 117, 1148-1150. Ou W.-J., Ito A., Okazaki H. and Omura T. (1989) Purification and characterization of a processing protease from rat liver mitochondria. EMBO Journal 8, 26052612. Schulte U., Arretz M., Schneider H., Tropschug M., Wachter E., Neupert W. and Weiss H. (1989) A family of mitochondrial proteins involved in bioenergetics and biogenesis. Nature 339, 147-149. Yang M., Jensen R. E., Yaffe M. P., Oppliger W. and Schatz G. (1988) Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS 1 and MAS 2 genes. EMBO Journal 7, 3857-3862.