The mitochondrial IMP peptidase of yeast: functional analysis of domains and identification of Gut2 as a new natural substrate

The mitochondrial inner membrane peptidase IMP of Saccharomyces cerevisiae is required for proteolytic processing of certain mitochondrially and nucleus-encoded proteins during their export from the matrix into the inner membrane or the intermembrane space. The membrane-associated signal peptidase complex is composed of the two catalytic subunits, Imp1 and Imp2, and the Som1 protein. The IMP subunits are thought to function in membrane association, interaction and stabilisation of subunits, substrate specificity, and proteolysis. We have analysed inner membrane peptidase mutants and substrates to gain more insight into the functions of various domains and investigate the basis of substrate recognition. The results suggest that certain conserved glycine residues in the second and third conserved regions of Imp1 and Imp2 are important for stabilisation of the Imp complex and for the proteolytic activity of the subunits, respectively. The non-conserved C-terminal parts of the Imp subunits are important for their proteolytic activities. The C-terminal region of Imp2, comprising a predicted second transmembrane segment, is dispensable for the stability of Imp2 and Imp1, and cannot functionally substitute for the C-terminal segment of Imp1. Alteration of the Imp2 cleavage site in cytochrome c ₁ (from AM to ND) reveals the specificity of the Imp2 peptidase. In addition, we have identified Gut2, the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, as a new substrate for Imp1. Failure to cleave the Gut2 precursor may contribute to the pet phenotype of certain imp mutants. Gut2 is associated with the inner membrane, and is essential for growth on glycerol-containing medium. Suggested functions of the analysed residues and domains of the IMP subunits, characteristics of the cleavage sites of substrates and implications for the phenotypes of imp mutants are discussed.Communicated by C. P. Hollenberg     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28°?C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.     

Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase

Summary The nuclear yeast mutant pet ts2858 is defective in the removal of pre-sequences from the mitochondrially encoded cytochrome oxidase subunit II (COXII) and the processing intermediate of cytochrome b ₂ (Cytb ₂), a nuclear gene product. In order to identify the genetic lesion in this mutant we have cloned and characterized a DNA region which complements the pet ts2858 mutation. The DNA sequence revealed three open reading frames, one of which is responsible for the complementation. A 570 by reading frame represents the structural gene PET2858, as demonstrated by in vitro mutagenesis, gene expression from a foreign promoter, and allelism tests. PET2858 encodes a 21.4 kDa protein, which is essential for growth on non-fermentable carbon sources and for the proteolytic processing of COXII and the Cytb ₂ intermediate. When the N-terminus of the PET2858 protein is fused to a reporter protein, the resulting hybrid molecule is imported into mitochondria. Interestingly, the N-terminal half of the deduced PET2858 protein exhibits 30.7% amino acid identity to the leader peptidase of Escherichia coli. These results suggest that PET2858 codes for a mitochondrial inner membrane protease (IMP1) or at least a subunit of it. This protease is involved in protein processing and export from the mitochondrial matrix.Dedicated to Professor Dr. Peter Starlinger on the occasion of his 60th birthday     

Cargo sequences are important for Som1p-dependent signal peptide cleavage in yeast mitochondria

The inner membrane protease (IMP) has two catalytic subunits, Imp1p and Imp2p, that exhibit nonoverlapping substrate specificity in mitochondria of the yeast Saccharomyces cerevisiae. The IMP also has at least one noncatalytic subunit, Som1p, which is required to cleave signal peptides from a subset of Imp1p substrates. To understand how Som1p mediates Imp1p substrate specificity, we addressed the possibility that Som1p functions as a molecular chaperone, which binds to specific substrates and directs them to the catalytic site. Our results show that cargo sequences attached to the signal peptide are important for Som1p-dependent presequence cleavage; however, no specific cargo sequence is required. Indeed, we show that a substrate normally destined for Imp2p is cleaved in a Som1p-dependent manner when the substrate is directed to Imp1p. These results argue against the notion that Som1p is a molecular chaperone. Instead, we propose that the cargo of some Imp1p substrates can assume a conformation incompatible with presequence cleavage. Som1p could thus act through Imp1p to improve cleavage efficiency early during substrate maturation.     

Substrate specificity of inner membrane peptidase in yeast mitochondria

The inner membrane protease (IMP) cleaves intra-organelle sorting peptides from precursor proteins in mitochondria of the yeast Saccharomyces cerevisiae. An unusual feature of the IMP is the presence of two catalytic subunits, Imp1p and Imp2p, which recognize distinct substrate sets even though both enzymes belong to the same protease family. This nonoverlapping substrate specificity was hypothesized to result from the recognition of distinct residues at the P′₁ position (also termed +1 position) in the protease substrates. Here, we constructed an extensive series of mutations to obtain a profile of the critical cleavage site residues in IMP substrates and conclude that Imp1p, and not Imp2p, recognizes specific P′₁ residues. In addition to its specificity for P′₁ residues, Imp1p also shows substrate specificity for the P₃ (−3) position. In contrast, Imp2p recognizes the P₁ (−1) position and the P₃ position. Based on this new understanding of IMP substrate specificity, we conducted a survey for candidate IMP substrates in mammalian mitochondria and found consensus Imp2p cleavage sites in mammalian precursors to cytochrome c₁ and glycerol-3-phosphate (G-3-P) dehydrogenase. Presence of a putative Imp2p cleavage site in G-3-P dehydrogenase was surprising, as its yeast ortholog contains an Imp1p cleavage site. To address this issue experimentally, we performed the first co-expression of mammalian IMP with proposed mammalian IMP precursors in yeast and show that murine precursors to cytochrome c₁ and G-3-P dehydrogenase are cleaved by murine Imp2p. These results suggest, surprisingly, G-3-P dehydrogenase has switched from Imp1p in yeast to Imp2p in mammals.     

A viable hypomorphic allele of the essential IMP3 gene reveals novel protein functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis.     

Imp3 unfolds stem structures in pre-rRNA and U3 snoRNA to form a duplex essential for small subunit processing

Eukaryotic ribosome biogenesis requires rapid hybridization between the U3 snoRNA and the pre-rRNA to direct cleavages at the A₀, A₁, and A₂ sites in pre-rRNA that liberate the small subunit precursor. The bases involved in hybridization of one of the three duplexes that U3 makes with pre-rRNA, designated the U3-18S duplex, are buried in conserved structures: box A/A′ stem–loop in U3 snoRNA and helix 1 (H1) in the 18S region of the pre-rRNA. These conserved structures must be unfolded to permit the necessary hybridization. Previously, we reported that Imp3 and Imp4 promote U3-18S hybridization in vitro, but the mechanism by which these proteins facilitate U3-18S duplex formation remained unclear. Here, we directly addressed this question by probing base accessibility with chemical modification and backbone accessibility with ribonuclease activity of U3 and pre-rRNA fragments that mimic the secondary structure observed in vivo. Our results demonstrate that U3-18S hybridization requires only Imp3. Binding to each RNA by Imp3 provides sufficient energy to unfold both the 18S H1 and the U3 box A/A′ stem structures. The Imp3 unfolding activity also increases accessibility at the U3-dependent A₀ and A₁ sites, perhaps signaling cleavage at these sites to generate the 5′ mature end of 18S. Imp4 destabilizes the U3-18S duplex to aid U3 release, thus differentiating the roles of these proteins. Protein-dependent unfolding of these structures may serve as a switch to block U3-pre-rRNA interactions until recruitment of Imp3, thereby preventing premature and inaccurate U3-dependent pre-rRNA cleavage and folding events in eukaryotic ribosome biogenesis.     

Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells

Background  

Pertussis toxin (PT) is an exotoxin virulence factor produced by Bordetella pertussis, the causative agent of whooping cough. PT consists of an active subunit (S1) that ADP-ribosylates the alpha subunit of several mammalian G proteins, and a B oligomer (S2–S5) that binds glycoconjugate receptors on cells. PT appears to enter cells by endocytosis, and retrograde transport through the Golgi apparatus may be important for its cytotoxicity. A previous study demonstrated that proteolytic processing of S1 occurs after PT enters mammalian cells. We sought to determine whether this proteolytic processing of S1 is necessary for PT cytotoxicity.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn₄Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme–substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.     

Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad. Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position. To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2). The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p. The remaining mutant signal peptides are cleaved inefficiently or not at all. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.     

The boxing glove shape of subunit <Emphasis Type="Italic">d</Emphasis> of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein

The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d _11–345, d _38–345, d _1–328 and d _1–298) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d _11–345, containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V₁V_O ATPase.     

Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.     

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G₂/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1–252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein–protein interactions as judged by in vitro pull-down assays. Direct protein–protein interactions were also observed between Pol1p–Imp4p, Pol1p–Sir4p and Sir4p–Zds2p, whereas no interaction was detected between Imp4p–Sir4p and Zds2p–Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1–252 region of Cdc13p was also analyzed. Expressing Cdc13(252–924)p, which lacks amino acids 1–252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G₂/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1–252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1–252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.     

High-light-induced superoxide anion radical formation in cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O₂ ^·−) in the cytochrome b ₆ f complex (Cyt b ₆ f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O₂ ^·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN₃ and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O₂ ^·−, which is produced under high-light illumination of the Cyt b ₆ f from spinach, might be generated from a reaction involing ¹O₂, and the Rieske Fe-S protein could serve as the electron donor in the O₂ ^·− production. The mechanism of photoprotection of the Cyt b ₆ f complex by antioxidants is discussed.     

F-box Protein Arabidillo-1 Promotes Lateral Root Development by Depressing the Functioning of GA<Subscript>3</Subscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, Arabidillo-1 and Arabidillo-2 have great sequence homology to Dictyostelium and metazoan β–catenin/Armadillo, which are important to animal and Dictyostelium development. Arabidillo-1 and Arabidillo-2 promote lateral root formation redundantly in Arabidopsis. Here, we showed that gibberellins (GA₃) has a greater inhibitory effect on lateral root growth from the null mutant arabidillo-1 than from the wild type, suggesting that the mechanism for Arabidillo-1-regulated modulation of lateral root proliferation is associated with GA₃-metabolic or signaling pathways. Our yeast two-hybrid analysis demonstrated that Arabidillo-1 interacts with ASK2 and ASK11, and that ASK2 can bind with the F-box domain of Arabidillo-1. Therefore, Arabidillo-1 is involved in the ubiquitin/26S proteasome-mediated proteolytic pathway. Based on these results, we conclude that Arabidillo-1 can degrade some positive regulator of the GA₃ signaling pathway through selective protein degradation of ubiquitin/26S. Moreover, that process is believed to be the mechanism for Arabidillo-1 promotion of lateral root development in Arabidopsis.