Complex protein targeting to dinoflagellate plastids

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.     

Targeting of tail‐anchored membrane proteins to subcellular organelles in Toxoplasma gondii

Proper protein localization is essential for critical cellular processes, including vesicle‐mediated transport and protein translocation. Tail‐anchored (TA) proteins are integrated into organellar membranes via the C‐terminus, orienting the N‐terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C‐terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early‐branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow‐up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C‐terminal sequence.      

Signal recognition particle mediates post-translational targeting in eukaryotes

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.     

THE ROLE OF CARBONIC ANHYDRASE IN THE FORMATION OF MOUGEOTIA (CHAROPHYCEAE) BLOOMS IN ACIDIC ENVIRONMENTS

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear-encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear-encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide-like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.     

IN VIVO CHARACTERIZATION OF THE DIATOM MULTIPARTATE PLASTID TARGETING SIGNAL

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear‐encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear‐encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide‐like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.     

Construction of system for localization of target protein in yeast periplasm using invertase

We constructed a novel system for periplasmic localization of target proteins, using yeast external invertase (INV) as anchor protein, in which the C- or N-terminal of the target protein was fused to the invertase and the fusion proteins expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH). Unlike in conventional cell-surface display, the system enables the target fusion protein to localize in yeast periplasm in a free state. As a model, enhanced green fluorescence protein (EGFP) was localized in yeast periplasm using the new system. Yeast-periplasm localization of INV-EGFP and EGFP-INV fusion proteins was confirmed by fluorescence microscopy and immunoblotting: green fluorescence was observed at the cell outline and, in western blot analysis, most fusion proteins were detected in the cell-surface fraction, indicating that the fusion proteins had been transported to the cell-surface layer. In addition, in both C- and N-terminal fusion, invertase showed activity, indicating dimer formation. These results demonstrate that invertase is a useful anchor for localizing target protein in the yeast periplasm.     

Analysis of tail-anchored protein translocation pathway in plants

Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants.     

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Internal plastid-targeting signal found in a RubisCO small subunit protein of a chlorarachniophyte alga

In all plants and algae, most plastid proteins are encoded by the nuclear genome and, consequently, need to be transported into plastids across multiple membranes. In organisms with secondary plastids, which evolved by secondary endosymbioses, and are surrounded by three or four envelope membranes, precursors of nuclear-encoded plastid proteins generally have an N-terminal bipartite targeting sequence that consists of an endoplasmic reticulum (ER)-targeting signal peptide (SP) and a transit peptide-like (TPL) sequence. The bipartite targeting sequences have been demonstrated to be necessary and sufficient for targeting proteins into the plastids of many algal groups, including chlorarachniophytes. Here, we report a new type of targeting signal that is required for delivering a RubisCO small subunit (RbcS) protein into the secondary plastids of chlorarachniophyte algae. In this study, we analyzed the plastid-targeting ability of an RbcS pre-protein, using green fluorescent protein (GFP) as a reporter molecule in chlorarachniophyte cells. We demonstrate that the N-terminal bipartite targeting sequence of the RbcS pre-protein is not sufficient, and that a part of the mature protein is also necessary for plastid targeting. By deletion analyses of amino acids, we determined the approximate location of an internal plastid-targeting signal within the mature protein, which is involved in targeting the protein from the ER into the chlorarachniophyte plastids.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

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.     

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.     

Is Protein Import into Plastids with Four‐Membrane Envelopes Dependent on Two Toc Systems Operating in Tandem?

Abstract: Plastids with four‐membrane envelopes have evolved by several independent endosymbioses involving a eukaryotic alga as the endosymbiont and a protozoan predator as the host. It is assumed that their outermost membrane is derived from the phagosomal membrane of the host and that protein targeting to and across this membrane proceeds co‐translationally, including ER and the Golgi apparatus (e.g., chlorarachniophytes) or only ER (e.g., heterokonts). Since the two inner membranes (or the plastid envelope) of such a complex plastid are derived from the endosymbiont plastid, they are probably provided with Toc and Tic systems, enabling post‐translational passage of the imported proteins into the stroma. The third envelope membrane, or the periplastid one, originates from the endosymbiont plasmalemma, but what import apparatus operates in it remains enigmatic. Recently, Cavalier‐Smith (1999^[5]) has proposed that the Toc system, pre‐existing in the endosymbiont plastid, has been relocated to the periplastid membrane, and that plastids having four envelope membranes contain two Toc systems operating in tandem and requiring the same targeting sequence, i.e., the transit peptide. Although this model is parsimonious, it encounters several serious obstacles, the most serious one resulting from the complex biogenesis of Toc75 which forms a translocation pore. In contrast to most proteins targeted to the outer membrane of the plastid envelope, this protein carries a complex transit peptide, indicating that a successful integration of the Toc system into the periplastid membrane would have to be accompanied by relocation of the stromal processing peptidase to the endosymbiont cytosol. However, such a relocation would be catastrophic because this enzyme would cleave the transit peptide off all plastid‐destined proteins, thus disabling biogenesis of the plastid compartment. Considering these difficulties, I suggest that in periplastid membranes two Toc‐independent translocation apparatuses have evolved: a porin‐like channel in chlorarachniophytes and cryptophytes, and a vesicular pathway in heterokonts and haptophytes. Since simultaneous evolution of a new transport system in the periplastid membrane and in the phagosomal one would be complicated, it is argued that plastids with four‐membrane envelopes have evolved by replacement of plastids with three‐membrane envelopes. I suggest that during the first round of secondary endosymbioses (resulting in plastids surrounded by three membranes), myzocytotically‐engulfed eukaryotic alga developed a Golgi‐mediated targeting pathway which was added to the Toc/Tic‐based apparatus of the endosymbiont plastid. During the second round of secondary endosymbioses (resulting in plastids surrounded by four membranes), phagocytotically‐engulfed eukaryotic alga exploited the Golgi pathway of the original plastid, and a new translocation system had to originate only in the periplastid membrane, although its emergence probably resulted in modification of the import machinery pre‐existing in the endosymbiont plastid.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids

Several groups of algae evolved by secondary endocytobiosis, which is defined as the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history such algae possess plastids that are surrounded by either three or four membranes. Protein targeting into plastids of these organisms depends on N-terminal bipartite presequences consisting of a signal and a transit peptide domain. This suggests that different protein targeting systems may have been combined during establishment of secondary endocytobiosis to enable the transport of proteins into the plastids. Here we demonstrate the presence of an apparently new type of transport into diatom plastids. We analyzed protein targeting into the plastids of diatoms and identified a conserved amino acid sequence motif within plastid preprotein targeting sequences. We expressed several diatom plastid presequence:GFP fusion proteins with or without modifications within that motif in the diatom Phaeodactylum tricornutum and found that a single conserved phenylalanine is crucial for protein transport into the diatom plastids in vivo, thus indicating the presence of a so far unknown new type of targeting signal. We also provide experimental data about the minimal requirements of a diatom plastid targeting presequence and demonstrate that the signal peptides of plastid preproteins and of endoplasmic reticulum-targeted preproteins in diatoms are functionally equivalent. Furthermore we show that treatment of the cells with Brefeldin A arrests protein transport into the diatom plastids suggesting that a vesicular transport step within the plastid membranes may occur.     

Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue

Efficient delivery of membrane proteins is a critical cellular process. The recently elucidated GET (Guided Entry of TA proteins) pathway is responsible for the targeted delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum. The central player is the ATPase Get3, which in its free form exists as a dimer. Biochemical evidence suggests a role for a tetramer of Get3. Here, we present the first crystal structure of an archaeal Get3 homologue that exists as a tetramer and is capable of TA protein binding. The tetramer generates a hydrophobic chamber that we propose binds the TA protein. We use small-angle X-ray scattering to provide the first structural information of a fungal Get3/TA protein complex showing that the overall molecular envelope is consistent with the archaeal tetramer structure. Moreover, we show that this fungal tetramer complex is capable of TA insertion. This allows us to suggest a model where a tetramer of Get3 sequesters a TA protein during targeting to the membrane.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Protein Transport in Plant Cells

The subcellular localization and secretion of proteins synthesized in the cytosol are determined by short amino acid sequences in their molecules. N-terminal transit peptides provide for protein translocation across the membranes of the ER, mitochondria, plastids, and microbodies. Later, these peptides are cleaved off by processing peptidases. C-terminal peptides direct some proteins into microbodies and vacuoles. Transport into the nucleus and insertion in the membranes are determined by the specific sequences that reside in the molecule of the mature protein. Specific receptors associated with the protein-translocating channel recognize transit peptides. Protein unfolding is required for successful protein transport through these channels. Chaperones maintain proteins in such a state. Folded proteins cross the nuclear pore complex and the membrane of microbodies. Protein transport is tightly associated with their processing. During the vesicular protein transport within the endomembrane system (ER, Golgi apparatus, plasma membrane, and vacuoles), correct protein targeting is ensured by protein sorting during vesicle loading, the assembly of corresponding protein coats, vesicle transport to the acceptor membrane, and specific membrane fusion.