Multiple pathways for the targeting of thylakoid proteins in chloroplasts

The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised proteins and their correct targeting into or across the thylakoid membrane. Biochemical and genetic studies have revealed the operation of several targeting pathways for these proteins, some of which are used for thylakoid lumen proteins whereas others are utilised by membrane proteins. Some pathways can be traced back to the prokarytoic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus. In this article we review recent findings in this field that point to the operation of a mechanistically unique protein translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor proteins.     

Targeting of lumenal proteins across the thylakoid membrane

The biogenesis of the plant thylakoid network is an enormously complex process in terms of protein targeting. The membrane system contains a large number of proteins, some of which are synthesized within the organelle, while many others are imported from the cytosol. Studies in recent years have shown that the targeting of imported proteins into and across the thylakoid membrane is particularly complex, with four different targeting pathways identified to date. Two of these are used to target membrane proteins: a signal recognition particle (SRP)-dependent pathway and a highly unusual pathway that appears to require none of the known targeting apparatus. Two further pathways are used to translocate lumenal proteins across the thylakoid membrane from the stroma and, again, the two pathways differ dramatically from each other. One is a Sec-type pathway, in which ATP hydrolysis by SecA drives the transport of the substrate protein through the membrane in an unfolded conformation. The other is the twin-arginine translocation (Tat) pathway, where substrate proteins are transported in a folded state using a unique mechanism that harnesses the proton motive force across the thylakoid membrane. This article reviews progress in studies on the targeting of lumenal proteins, with reference to the mechanisms involved, their evolution from endosymbiotic progenitors of the chloroplast, and possible elements of regulation.     

Fidelity of organellar protein targeting

The majority of cellular proteins are targeted to organelles. Cytosolic ribosomes produce these proteins as precursors with cleavable or non-cleavable targeting sequences that direct them to receptor proteins on the organelle surface. Multiple targeting factors ensure cellular sorting of the precursor proteins. In co-translational protein import, the ribosome-nascent chain complex is transported to the organellar protein translocase to couple protein synthesis and protein import. In post-translational mode, targeting factors like molecular chaperones guide the precursor proteins from ribosomes to the cell organelle. Defects in protein targeting and import cause mistargeting of proteins to different cellular compartments and challenge the balance of cellular proteostasis. Specific dislocases and degradation machineries remove such mislocalized proteins from the membrane to allow retargeting or their proteasomal turnover. In this review, we discuss targeting and quality control factors that ensure fidelity of protein targeting to mitochondria.     

A thioredoxin family protein of the apicoplast periphery identifies abundant candidate transport vesicles in Toxoplasma gondii

Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the apicoplast, is bounded by multiple membranes. In this report we identify a novel apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the apicoplast intermembrane spaces. ATrx1 lacks the apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the apicoplast.     

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.     

Presence of a latent mitochondrial targeting signal in gene on mitochondrial genome

Organelles, such as mitochondria and chloroplasts, are derived from endosymbionts. Gene transfer events from organelles to the nucleus have occurred over evolutionary time. In the case that a transferred gene in the nucleus needs to go back to the original organelle, it must obtain targeting information for sorting its protein to that organelle. Here, we reveal that the genes for the ribosomal proteins L2 and S4 in the Arabidopsis thaliana mitochondrial (mt) genome contain information for protein targeting into the mitochondria. Similarly, the genes for the ribosomal proteins L2 and S19 in the Oryza sativa mt genome contain information for protein targeting into mitochondria. These results suggest that targeting information already existed in each gene in the plant mt genome before the transfer event to the nucleus occurred. We provide new insights into the timing of the appearance of targeting signals in evolution.     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Life and death of a BiP substrate

BiP is the mammalian endoplasmic reticulum (ER) Hsp70 orthologue that plays a major role in all functions of this organelle including the seemingly opposing functions of aiding the maturation of unfolded nascent proteins and identifying and targeting chronically unfolded proteins for degradation. The recent identification of mammalian BiP co-factors combined with delineation of the ER degradation machinery and data suggesting that the ER is subdivided into unique regions helps explain how these different functions can occur in the same organelle and raises some unresolved issues.     

AP-1 in Toxoplasma gondii mediates biogenesis of the rhoptry secretory organelle from a post-Golgi compartment

We have previously demonstrated that Toxoplasma gondii has a tyrosine-based sorting system, which mediates protein targeting to the lysosome-like rhoptry secretory organelle. We now show that rhoptry protein targeting is also dependent on a dileucine motif and occurs from a post-Golgi endocytic organelle to mature rhoptries in an adaptin-dependent fashion. The T. gondii AP-1 adaptin complex is implicated in this transport because the micro1 chain of T. gondii AP-1 (a) was localized to multivesicular endosomes and the limiting and luminal membranes of the rhoptries; (b) bound to endocytic tyrosine motifs in rhoptry proteins, but not in proteins from dense granule secretory organelles; (c) when mutated in predicted tyrosine-binding motifs, led to accumulation of the rhoptry protein ROP2 in a post-Golgi multivesicular compartment; and (d) when depleted via antisense mRNA, resulted in accumulation of multivesicular endosomes and immature rhoptries. These are the first results to implicate AP-1 in transport from a post-Golgi compartment to a mature secretory organelle and substantially expand the role for AP-1 in anterograde protein transport.     

Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex. We identify Tom34 as a cochaperone of Hsp70/Hsp90 in mitochondrial protein import. We show that Tom34 is an integral component with Hsp70 and Hsp90 in the large complex. We also demonstrate the role of Tom34 in the mitochondrial import process, as the addition of an excess of Tom34 prevents efficient mitochondrial translocation of precursor proteins that have requirements for Hsp70/Hsp90. Tom34 exhibits an affinity for mitochondrial preproteins of the Tom70 translocation pathway as demonstrated by binding assays using in vitro translated proteins as baits. In addition, we examined the specificity and the size of different complex cytosolic machines. Separation of different radiolabeled cell-free translated proteins on Native-PAGE showed the presence of a high molecular weight complex which binds hydrophobic proteins. Importantly we show that the formation of the chaperone cytosolic complex that mediates the targeting of proteins to the mitochondria contains Tom34 and assembles in the presence of a fully translated substrate protein.     

Dual targeting to mitochondria and chloroplasts.

Plant cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids. Their endosymbiotic origin explains why these organelles contain their own DNA, nonetheless only a few dozens of genes are actually encoded by these genomes. Many of the other genes originally present have been transferred to the nuclear genome of the host, the product of their expression being targeted back to the corresponding organelle. Although targeting of proteins to mitochondria and chloroplasts is generally highly specific, an increasing number of examples have been discovered where the same protein is imported into both organelles. The object of this review is to compare and discuss these examples in order to try and identify common features of dual-targeted proteins. The study helps throw some light on the factors determining organelle targeting specificity, and suggests that dual-targeted proteins may well be far more common than once thought.     

Protein targeting to the malaria parasite plastid

The relict plastid, or apicoplast, of the malaria parasite Plasmodium falciparum is an essential organelle and a promising drug target. Most apicoplast proteins are nuclear encoded and post-translationally targeted into the organelle using a bipartite N-terminal extension, consisting of a typical endomembrane signal peptide and a plant-like transit peptide. Apicoplast protein targeting commences through the parasite's secretory pathway. We review recent experimental evidence suggesting that the apicoplast resides in the mainstream endomembrane system proximal to the Golgi. Further, we explore possible mechanisms for translocation of nuclear-encoded apicoplast proteins across the four bounding membranes. Recent insights into the composition of the transit peptide and how it is cleaved and degraded after use are also examined. Characterization of apicoplast targeting has not only shed light on how this group of parasites mediate intracellular protein trafficking events but also it has helped identify new targets for therapeutics. The distinctive leader sequences of apicoplast proteins make them readily identifiable, allowing assembly of a virtual organelle metabolome from the genome. Such analysis has lead to the identification of several biochemical pathways that are absent from the human host and thus represent novel therapeutic targets for parasitic infection.     

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.     

Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.     

The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria

Peroxisomes and mitochondria are multifunctional eukaryotic organelles that are not only interconnected metabolically but also share proteins in division. Two evolutionarily conserved division factors, dynamin-related protein (DRP) and its organelle anchor FISSION1 (FIS1), mediate the fission of both peroxisomes and mitochondria. Here, we identified and characterized a plant-specific protein shared by these two types of organelles. The Arabidopsis thaliana PEROXISOMAL and MITOCHONDRIAL DIVISION FACTOR1 (PMD1) is a coiled-coil protein tethered to the membranes of peroxisomes and mitochondria by its C terminus. Null mutants of PMD1 contain enlarged peroxisomes and elongated mitochondria, and plants overexpressing PMD1 have an increased number of these organelles that are smaller in size and often aggregated. PMD1 lacks physical interaction with the known division proteins DRP3 and FIS1; it is also not required for DRP3's organelle targeting. Affinity purifications pulled down PMD1's homolog, PMD2, which exclusively targets to mitochondria and plays a specific role in mitochondrial morphogenesis. PMD1 and PMD2 can form homo- and heterocomplexes. Organelle targeting signals reside in the C termini of these proteins. Our results suggest that PMD1 facilitates peroxisomal and mitochondrial proliferation in a FIS1/DRP3-independent manner and that the homologous proteins PMD1 and PMD2 perform nonredundant functions in organelle morphogenesis.     

Import of stably folded proteins into peroxisomes.

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

Signalling to the nucleus via A-kinase anchoring proteins

The cell nucleus is a highly dynamic organelle whose function and structure during the cell cycle is tightly controlled. A number of signals triggered by external stimuli or intracellular clocks are relayed to the nucleus by protein kinases and phosphatases. Specificity of action of kinases and phosphatases can be achieved by their recruitment into multiprotein complexes targeted to discrete subcellular or subnuclear loci. One class of molecules targeting signalling units within single complexes are A-kinase anchoring proteins or AKAPs. AKAPs not only target enzymes to their substrate but may also regulate enzyme activity. This chapter highlights the role of nuclear AKAPs in relaying and modulating protein kinase and phosphatase signals to the nucleus or chromosomes.