Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.     

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction.     

Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting

Background information. Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule‐regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3′/RB3″ are localized to the Golgi and vesicle‐like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. Results. We show that their conserved N‐terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle‐like localization. Using palmitoylation‐deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co‐operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin‐like domain) extension. Conclusions. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.     

Altered hepatic mitochondrial ribosome structure following chronic ethanol consumption.

Chronic ethanol consumption decreases the synthesis of all 13 polypeptides encoded by the hepatic mitochondrial genome. This alteration in mitochondrial protein synthesis is due to modifications in mitochondrial ribosomes. In the current study, the nature of these alterations was investigated by determining some of the hydrodynamic properties, namely sedimentation coefficient, shape, and mass of mitochondrial ribosomes. The effect of ethanol consumption on the capacity for mitochondrial ribosomes to translate proteins was also determined using an in vitro Poly (U) assay system. Rats were fed the Lieber-DeCarli diet for 31 days with ethanol as 36% of total calories. The sedimentation coefficient, measured by sedimentation velocity analyses, was slightly, but significantly lower in ethanol mitochondrial ribosomes (53.2 +/- 0.5S) when compared with pair-fed controls (54.1 +/- 0.5S) (P = 0.04). Mitochondrial ribosomes from ethanol-fed animals also had a greater tendency to dissociate into subunits. The diffusion coefficient, determined by dynamic light scattering, was lower in mitochondrial ribosomes from ethanol-fed rats than pair-fed controls and this indicated a significantly greater diameter for ethanol ribosomes (42.1 +/- 0.2 nm) than for preparations from pair-fed controls (39.1 +/- 0.5 nm; P = 0.008). These alterations to ethanol mitochondrial ribosomes occurred despite no change in molecular mass, which suggested a significant ethanol-related shape change in the ribosomes. The translation capacity of mitochondrial ribosome preparations from ethanol-fed animals was markedly reduced due to dissociation of the monosome into light and heavy subunits. In summary, these observations demonstrate that chronic ethanol consumption causes significant structural and functional alterations to mitochondrial ribosomes. The loss in ribosome function leads to impaired mitochondrial polypeptide synthesis and is an example of a pathology giving rise to an alteration in the mitochondrial ribosome structure.     

A structural model for the large subunit of the mammalian mitochondrial ribosome

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.     

Bacterial proteins predisposed for targeting to mitochondria

Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle. According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont. We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria. This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.     

The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway

1. Download : Download high-res image (130KB)
2. Download : Download full-size image

     

Analysis of dinoflagellate mitochondrial protein sorting signals indicates a highly stable protein targeting system across eukaryotic diversity

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.     

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.     

Feature-extraction from endopeptidase cleavage sites in mitochondrial targeting peptides

Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps. Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions −10, −3, and −2. A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs. Neural networks were used to define local sequence features around precursor cleavage sites. Proteins 30:49–60, 1998. © 1998 Wiley-Liss, Inc.     

A pathway for the transmission of allosteric signals in the ribosome through a network of RNA tertiary interactions

There are a large number of tertiary contacts between nucleotides in 23S rRNA, but which are of functional importance is not known. Disruption of one between A2662 in the sarcin/ricin loop (SRL) and A2531 in the peptidyl-transferase center (PTC) has adverse effects on cell growth and on the ability of ribosomes to catalyze some but not other partial reactions of elongation. A lethal A2662C mutation is suppressed by a concomitant lethal A2531 mutation. Ribosomes with non-lethal A2531 mutations, treated with base-specific reagents, have alterations of nucleotides in the PTC (home of A2531) and, more significantly, in nucleotides in the SRL and in the GTPase center. The results suggest that the function of ribosomal centers is coordinated by a set of sequential conformational changes in rRNA that are a response to signals transmitted through a network of tertiary interactions.     

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal-anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.     

The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon

Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SRβ subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SRβ subunit and how they co-ordinate RNC transfer is unknown. By site-directed cross-linking and fluorescence resonance energy transfer (FRET) analyses, we show that FtsY, the bacterial SRα homologue, binds to the exposed C4/C5 loops of SecY, the central component of the bacterial Sec translocon. The same loops serve also as binding sites for SecA and the ribosome. The FtsY-SecY interaction involves at least the A domain of FtsY, which attributes an important function to this so far ill-defined domain. Binding of FtsY to SecY residues, which are also used by SecA and the ribosome, probably allows FtsY to sense an available translocon and to align the incoming SRP-RNC with the protein conducting channel. Thus, the Escherichia coli FtsY encompasses the functions of both the eukaryotic SRα and SRβ subunits in one single protein.     

Cancer — the mitochondrial connection

Mitochondria are the powerhouse of the cell. They play a vital role in the energy metabolism and regulate calcium flux and apoptosis. The recent resurgence of interest in mitochondrial studies is largely attributed to the recognition that mitochondrial dysfunctions lead to various physiopathological disorders, especially in the development and progression of cancer. Mitochondrial DNA is very susceptible to mutations, which lead to respiratory dysfunction and are implicated in many cancers. Mitochondria serve as the molecular target for a structurally diverse group of pharmacological agents in cancer chemotherapy. Biochemical and biophysical characterization have helped to identify several important differences between mitochondria of normal and disease state. Such unique alterations in mitochondrial structure and function could become promising target for the development of new generation drugs. The author has expired after preparation of this article. Dr. Anup K. Mukhopadhyay was Associate Professor, Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab — 160062, India.     

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.     

The polypeptide tunnel exit of the mitochondrial ribosome is tailored to meet the specific requirements of the organelle

The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co‐translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co‐factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially‐encoded proteins.     

Binding of a synthetic targeting peptide to a mitochondrial channel protein

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) fromNeurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochromec in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochromec bind to protein arms which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.     

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito‐C. Mito‐C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito‐C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF‐1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER–mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito‐C counteracts dengue virus‐induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito‐C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti‐viral research.     

Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane--a guided tour

Only a few mitochondrial proteins are encoded by the organellar genome. The majority of mitochondrial proteins are nuclear encoded and thus have to be transported into the organelle from the cytosol. Within the mitochondrion proteins have to be sorted into one of the four sub-compartments: the outer or inner membranes, the intermembrane space or the matrix. These processes are mediated by complex protein machineries within the different compartments that act alone or in concert with each other. The translocation machinery of the outer membrane is formed by a multi-subunit protein complex (TOM complex), that is built up by signal receptors and the general import pore (GIP). The inner membrane houses two multi-subunit protein complexes that each handles special subsets of mitochondrial proteins on their way to their final destination. According to their primary function these two complexes have been termed the pre-sequence translocase (or TIM23 complex) and the protein insertion complex (or TIM22 complex). The identification of components of these complexes and the analysis of the molecular mechanisms underlying their function are currently an exciting and fast developing field of molecular cell biology.     

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.