Dual Targeting Of Proteins Research Articles

Mitochondria, Peroxisomes and Beyond-How Dual Targeting Regulates Organelle Tethering.

Eukaryotic cells feature distinct membrane-enclosed organelles such as mitochondria and peroxisomes, each playing vital roles in cellular function and organization. These organelles are linked at membrane contact sites, facilitating interorganellar molecule and ion exchange. Most contact-forming proteins identified to date are membrane proteins or membrane-associated proteins, which can form very stable contacts. Recent findings suggest additional mechanistically distinct tethering events that arise from dual protein targeting. Proteins bearing targeting signals for multiple organelles, such as an N-terminal signal for mitochondria and a C-terminal signal for peroxisomes, function as tethers, fostering contacts by engaging targeting factors at both organelles. A number of dually targeted membrane proteins can contribute to contact site formation and transit from one organelle to the other as well. These interactions may enable the fine-tuning of organelle proximity, hence, adapting connections to meet varying physiological demands.

Rather rule than exception? How to evaluate the relevance of dual protein targeting to mitochondria and chloroplasts

Dual targeting of a nuclearly encoded protein into two different cell organelles is an exceptional event in eukaryotic cells. Yet, the frequency of such dual targeting is remarkably high in case of mitochondria and chloroplasts, the two endosymbiotic organelles of plant cells. In most instances, it is mediated by "ambiguous" transit peptides, which recognize both organelles as the target. A number of different approaches including in silico, in organello as well as both transient and stable in vivo assays are established to determine the targeting specificity of such transit peptides. In this review, we will describe and compare these approaches and discuss the potential role of this unusual targeting process. Furthermore, we will present a hypothetical scenario how dual targeting might have arisen during evolution.

'2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

We report the initial characterization of an N‐terminal oligopeptide ‘2A‐like’ sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A‐mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A‐like N‐terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A‐mediated translational recoding has occurred: the 2A‐like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A‐like signal sequence and is localized to the cytoplasm. This type of dual‐functional signal sequence results, therefore, in the partitioning of the translation products between the two sub‐cellular sites and represents a newly described form of dual protein targeting.

The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s) encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes.

133. Use of 2As To Control Protein Subcellular Localization

A substantial proportion (~39%) of all human proteins are either secreted from the cell, located within the lumen/ membranes of cytoplasmic vesicular structures, or, are plasma membrane proteins. Given that such high proportion of proteins are initially translocated into the endoplasmic reticulum (ER), many therapeutic strategies rely on the ability to co-express multiple proteins – some, or all of which, might be targeted to such sites. This directly applies to in vivo gene therapy strategies, or, when therapeutic proteins may need to be co-expressed with selectable markers (e.g. ex vivo gene therapies).As was shown before, Picornavirus 2A (foot-and-mouth disease virus 2A; F2A) and ‘2A-like’ sequences are powerful tools that allow multiple proteins to be translated and co-expressed from a single transcript mRNA under the control of only one promoter. When 2A is positioned between sequences encoding two, or more, genes, it mediates a co-translational ‘cleavage’ at its own C-terminus. A major problem with co-expression of certain proteins targeted to, or transiting through, the ER is that the ‘cleavage’ activity of short F2As can be greatly inhibited by sequences immediately upstream leading to aberrant sub-cellular localisation of some proteins.We have also discovered a number of active cellular 2A-like sequences, associated with non-long terminal repeat (non-LTR) retrotransposons, but also with structural and metabolic proteins: ankyrin repeats, sodium dependent neutral amino acid transporters, and NOD-like receptor (NLR) proteins. Examination of the surrounding protein and gene structure revealed that in the majority of cases these 2A sequences occurred as N-terminal features. Interestingly, using SignalP, many of these novel 2As scored highly as N-terminal signal peptides.Here, we present our latest findings on 2A sequences. A series of test proteins (eGFP and mCherry) were expressed i) followed by ‘hybrid’ ‘self-cleaving’ F2A sequences (with different upstream contexts) or ii) downstream of a putative signal 2A. We demonstrate that inhibition of F2A-mediated cleavage in shorter sequences can be overcome by introduction of mutations upstream of 2A changing the context of the sequence between the C-terminus of the upstream protein and 2A sequence. In the case of N-terminal – cellular – (NLR) 2As, ‘uncleaved’ 2A indeed can act as a signal peptide. If 2A does not ‘cleave’, it directs a proportion of the newly synthesised reporter protein to the exocytic pathway: if 2A ‘cleaves’, the protein downstream is localised to the cytoplasm. This type of 2A mediates, therefore, a newly discovered form of dual protein targeting.

Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in 15N HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal φχχφφ motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors.

Protein folding as a driving force for dual protein targeting in eukaryotes.

It is well documented that in eukaryotic cells molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized identical or nearly identical proteins are termed “echoforms.” Our conventional definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a membrane. Thus, dual targeted proteins are recognized by at least one organelle's receptors and translocation machineries within the lipid bilayer. In this review we attempt to evaluate mechanisms and situations in which protein folding is the major determinant of dual targeting and of the relative distribution levels of echoforms in the subcellular compartments of the eukaryotic cell. We show that the decisive folding step can occur prior, during or after translocation through the bilayer of a biological membrane. This phenomenon involves folding catalysts in the cell such as chaperones, proteases and modification enzymes, and targeting processes such as signal recognition, translocation through membranes, trapping, retrotranslocation and reverse translocation.

Widespread dual targeting of proteins in land plants: When, where, how and why

Since the discovery of the first dual targeted protein in plants in 1995 the number of dual targeted proteins in plants has grown to over 250 proteins. Much work and investigations have focused on identifying how or what makes a protein dual targeted. Recently, more research has focused on the evolution and conservation of dual targeting of proteins in plants. This new work has demonstrated that dual targeting arose early within the evolution of plants and because it is rarely lost, once gained, it must be under some positive selection pressure. The possible reasons as why proteins are dual targeted and why it was conserved during the evolution of plants are discussed.

Monotopic topology is required for lipid droplet targeting of ancient ubiquitous protein 1

Ancient ubiquitous protein 1 (AUP1) is a multifunctional protein, which acts on both lipid droplets (LDs) and the endoplasmic reticulum (ER) membrane. Double localization to these two organelles, featuring very different membrane characteristics, was observed also for several other integral proteins, but little is known about the signals and mechanisms behind dual protein targeting to ER and LDs. Here we dissect the AUP1 targeting signals by analyses of localization and topology of several deletion and point mutants. We found that AUP1 is inserted into the membrane of the ER in a monotopic hairpin fashion, and subsequently transported to the hemi-membrane of LDs. A single domain localized in the N-terminal part of AUP1 enables its ER residence, the monotopic insertion, and the LD localization. Different specific residues within this multifunctional domain are responsible for achieving the complex spatial distribution pattern. A mutation of three amino acids, which changes AUP1 topology from hairpin to transmembrane, abolishes LD localization. These findings suggest that the cell is able to target a protein to multiple intracellular locations using a single domain.

Dual targeting of peroxisomal proteins

Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed.

Evolutionary origins of metabolic compartmentalization in eukaryotes.

Many genes in eukaryotes are acquisitions from the free-living antecedents of chloroplasts and mitochondria. But there is no evolutionary 'homing device' that automatically directs the protein product of a transferred gene back to the organelle of its provenance. Instead, the products of genes acquired from endosymbionts can explore all targeting possibilities within the cell. They often replace pre-existing host genes, or even whole pathways. But the transfer of an enzymatic pathway from one compartment to another poses severe problems: over evolutionary time, the enzymes of the pathway acquire their targeting signals for the new compartment individually, not in unison. Until the whole pathway is established in the new compartment, newly routed individual enzymes are useless, and their genes will be lost through mutation. Here it is suggested that pathways attain novel compartmentation variants via a 'minor mistargeting' mechanism. If protein targeting in eukaryotic cells possesses enough imperfection such that small amounts of entire pathways continuously enter novel compartments, selectable units of biochemical function would exist in new compartments, and the genes could become selected. Dual-targeting of proteins is indeed very common within eukaryotic cells, suggesting that targeting variation required for this minor mistargeting mechanism to operate exists in nature.

Prediction of dual protein targeting to plant organelles

* Dual targeting of proteins to more than one subcellular localization has been found in animals, in fungi and in plants. In the latter, ambiguous N-terminal targeting signals have been described that result in the protein being located in both mitochondria and plastids. We have developed ambiguous targeting predictor (ATP), a machine-learning implementation that classifies such ambiguous targeting signals. * Ambiguous targeting predictor is based on a support vector machine implementation that makes use of 12 different amino acid features. Prediction results were validated using fluorescent protein fusion. * Both in silico and in vivo evaluations demonstrate that ambiguous targeting predictor is useful for predicting dual targeting to mitochondria and plastids. Proteins that are targeted to both organelles by tandemly arrayed signals (so-called twin targeting) can be predicted by both ambiguous targeting predictor and a combination of single targeting prediction tools. Comparison of ambiguous targeting predictor with previous experimental approaches, as well as in silico approaches, shows good congruence. * Based on the prediction results, land plant genomes are expected to encode, on average, > 400 proteins that are located in mitochondria and plastids. Ambiguous targeting predictor is helpful for functional genome annotation and can be used as a tool to further our understanding about dual protein targeting and its evolution.

Protein transport in organelles: Dual targeting of proteins to mitochondria and chloroplasts

As many as fifty proteins have now been experimentally demonstrated to be targeted to both mitochondria and plastids, a phenomenon referred to as dual targeting. Although the first reported case of dual targeting of a protein was reported in 1995, there is still little understanding of the mechanism of dual targeting and any similarities or differences with respect to the targeting of location-specific proteins. This minireview summarizes dual targeting in terms of signals, passenger proteins, receptors, regulation, why proteins may need to be dual targeted and the future challenges that remain in this area.

Single translation—dual destination: mechanisms of dual protein targeting in eukaryotes

It is well documented that single eukaryotic genes can give rise to proteins that are localized to several subcellular locations. This is achieved at the level of transcription, splicing and translation, and results in two or more translation products that either harbour or lack specific targeting signals. Nevertheless, the possibility of dual targeting of a single translation product has recently emerged. Here, we review cases of such dual targeting with emphasis on the mechanisms through which these phenomena occur. Proteins that harbour one signal, two separate signals or an overlapping ambiguous signal may follow dual distribution in the cell. The mechanism of dual targeting is driven by the competition or promiscuity of various molecular events. Protein folding, post-translational modification and protein-protein interaction are key players in this phenomenon.

The use of multiple transcription starts causes the dual targeting of Arabidopsis putative monodehydroascorbate reductase to both mitochondria and chloroplasts.

Monodehydroascorbate reductase (MDAR) isoforms exist in mitochondria, chloroplasts, cytosol and microbodies. Two putative MDAR sequences with an extended N-terminal region are found in Arabidopsis. They differ in the length of the extension by 21 bp. We have shown that these two isoforms arise from a single gene by the use of multiple transcription starts. Green fluorescent protein was fused to each extension, revealing that the longer and shorter fusion proteins were imported into mitochondria and chloroplasts, respectively. These results demonstrate that putative MDAR is a dual-targeting protein transported into both mitochondria and chloroplasts. Although there have been several reports of dual targeting of proteins to mitochondria and chloroplasts, this is the first example in which the dual targeting of the protein to mitochondria and chloroplasts is achieved by the use of multiple transcription initiation sites.