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.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

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.     

Import of proteins into mitochondria and chloroplasts

Although mitochondria and chloroplasts synthesize some of their own proteins, they must import most of them from the cytosol. Import is mediated by molecular chaperones in the cytosol, receptors and channels in the organelle membranes and ATP-driven 'import motors' inside the organelles. Many of these components are now known, allowing informed guesses on how they might work.     

Peroxisome biogenesis in yeast

Eukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location(s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle(s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes. Proteins carry cis-acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery. Sequences that target proteins to the endoplasmic reticulum/secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino-terminal extensions. In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post-translational modification. This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in yeast.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco

N-terminal presequences from cDNAs encoding mitochondrion- or chloroplast-specific proteins are able, with variable efficiencies, to target preproteins to their respective organelles. In the few cases studied in which a nuclear-encoded protein is found in both these organelles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondria and the cytosol. Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in both chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequence with presequences from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochondrion) resulted in targeting of GR only into the appropriate organelle. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acetyl transferase resulted in targeting of the foreign protein to chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and mitochondria in vivo . This is the first example of co-targeting by a higher plant preprotein.     

Policing Tic 'n' Toc, the doorway to chloroplasts

The organization of eukaryotic cells into different membrane-enclosed compartments requires an ordered and regulated system for targeting and translocating proteins synthesized in the cytosol across organellar membranes. Protein translocation through integral membrane proteinaceous complexes shares common principles in different organelles, whereas molecular mechanisms and energy requirements are diverse. Translocation into mitochondria and plastids requires most proteins to cross two membranes, and translocation must be regulated to accommodate environmental or metabolic changes. In the last decade, the first ideas were formulated about the regulation of protein translocation into chloroplasts, thereby laying the foundation for this field. Here, we describe recent models for the regulation of translocation by precursor protein phosphorylation, receptor dimerization, redox sensing and calcium signaling. We suggest how these mechanisms might fit within the regulatory framework for the entry of proteins into chloroplasts.     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

The ER protein Ema19 facilitates the degradation of nonimported mitochondrial precursor proteins

For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of nonproductive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homologue is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

An antimicrobial origin of transit peptides accounts for early endosymbiotic events

Primary endosymbiosis, which gave rise to mitochondria or chloroplasts, required successful targeting of a number of proteins from the host cytosol to the endosymbiotic organelles. A survey of studies published in separate fields of biological research over the past 40 years argues for an antimicrobial origin of targeting peptides. It is proposed that mitochondria and chloroplast derive from microbes that developed a resistance strategy to antimicrobial peptides that consisted in their rapid internalization and proteolytic disposal by microbial peptidases.      

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Quality Control: Proteins and Organelles

This review summarizes materials on the mechanisms of intracellular degradation of proteins whose topogenesis is disturbed at one stage or another. Chaperone and proteolytic systems involved in this process in the endoplasmic reticulum, mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct subcellular compartments of procaryotic cells are considered. The available data suggest that living cells contain numerous systems keeping under control both folding of newly synthesized and newly imported polypeptide chains and their incorporation into heterooligomeric complexes. The point of view is elaborated that organelle formation is controlled not only at the level of individual protein molecules but also at the supermolecular level when whole organelles incapable of carrying out their integral key functions become targets for partial or total elimination. This type of control is realized through an autophagic mechanism involving lysosomes/vacuoles.     

Cytosolic events involved in chloroplast protein targeting

Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.     

Structural features within the nascent chain regulate alternative targeting of secretory proteins to mitochondria

Protein targeting to specified cellular compartments is essential to maintain cell function and homeostasis. In eukaryotic cells, two major pathways rely on N‐terminal signal peptides to target proteins to either the endoplasmic reticulum (ER) or mitochondria. In this study, we show that the ER signal peptides of the prion protein‐like protein shadoo, the neuropeptide hormone somatostatin and the amyloid precursor protein have the property to mediate alternative targeting to mitochondria. Remarkably, the targeting direction of these signal peptides is determined by structural elements within the nascent chain. Each of the identified signal peptides promotes efficient ER import of nascent chains containing α‐helical domains, but targets unstructured polypeptides to mitochondria. Moreover, we observed that mitochondrial targeting by the ER signal peptides correlates inversely with ER import efficiency. When ER import is compromised, targeting to mitochondria is enhanced, whereas improving ER import efficiency decreases mitochondrial targeting. In conclusion, our study reveals a novel mechanism of dual targeting to either the ER or mitochondria that is mediated by structural features within the nascent chain.     

The surprising complexity of peroxisome biogenesis

Peroxisomes are small organelles with a single boundary membrane. All of their matrix proteins are nuclear-encoded, synthesized on free ribosomes in the cytosol, and post-translationally transported into the organelle. This may sound familiar, but in fact, peroxisome biogenesis is proving to be surprisingly unique. First, there are several classes of plant peroxisomes, each specialized for a different metabolic function and sequestering specific matrix enzymes. Second, although the mechanisms of peroxisomal protein import are conserved between the classes, multiple pathways of protein targeting and translocation have been defined. At least two different types of targeting signals direct proteins to the peroxisome matrix. The most common peroxisomal targeting signal is a tripeptide limited to the carboxyl terminus of the protein. Some peroxisomal proteins possess an amino-terminal signal which may be cleaved after import. Each targeting signal interacts with a different cytosolic receptor; other cytosolic factors or chaperones may also form a complex with the peroxisomal protein before it docks on the membrane. Peroxisomes have the unusual capacity to import proteins that are fully folded or assembled into oligomers. Although at least 20 proteins (mostly peroxins) are required for peroxisome biogenesis, the role of only a few of these have been determined. Future efforts will be directed towards an understanding of how these proteins interact and contribute to the complex process of protein import into peroxisomes.     

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail‐anchored proteins

Tail‐anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein‐free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5‐ER and b5‐RR, which differ only in the charge of the C‐terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell‐free and in cellula results, we analyzed targeting in digitonin‐permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C‐terminal negative charge of b5‐ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ES_I) reduced insertion of b5‐ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ES_I, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C‐terminal charge, and reveal a novel, substrate‐specific ER‐targeting pathway.      

A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.     

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins

Subcellular organelles in eukaryotes are surrounded by lipid membranes.In an endomembrane system,vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles.However,organellar proteins for chloroplasts,mitochondria,the nucleus,and peroxisomes that are translated in the cytosol are directly imported into their target organelles.Chloroplasts are a plant-specific organelle with outer and inner envelope membranes,a dual-membrane structure that is simil...