Protein–protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids

Most secondary plastids of red algal origin are surrounded by four membranes and nucleus‐encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD‐(ER‐associated degradation) derived machinery termed SELMA (symbiont‐specific ERAD‐like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein–protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD‐related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.     

Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The symbiont-derived ERAD-like machinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum. The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.     

New mechanistic insights into pre‐protein transport across the second outermost plastid membrane of diatoms

Chromalveolates like the diatom Phaeodactylum tricornutum arose through the uptake of a red alga by a phagotrophic protist, a process termed secondary endosymbiosis. In consequence, the plastids are surrounded by two additional membranes compared with primary plastids. This plastid morphology poses additional barriers for plastid‐destined proteins, which are mostly nucleus‐encoded. Recent investigations have focused on the postulated translocon of the second outermost membrane (periplastidal membrane, PPM). These studies identified a symbiont‐specific ERAD (endoplasmic reticulum‐associated degradation)‐like machinery (SELMA), which has been implicated in plastid pre‐protein import. Despite this recent progress, key factors for protein transport via SELMA are still unknown. As SELMA substrates presumably undergo ubiquitination, a corresponding ubiquitin ligase and an enzyme for the subsequent removal of ubiquitin need to reside in the space between the second and third membrane (periplastidal compartment, PPC). Here we characterize two proteins fulfilling these criteria. We show that ptE3P (P. t ricornutumE3 enzyme of the P PC), the ubiquitin ligase, and ptDUP (P. t ricornutumd e‐u biquitinating enzyme of the P PC), the de‐ubiquitinase, localize to the PPM and PPC, respectively. In addition, we demonstrate their retained functionality by in vitro data.     

Three old and one new: protein import into red algal-derived plastids surrounded by four membranes

Engulfment of a red or green alga by another eukaryote and subsequent reduction of the symbiont to an organelle, termed a complex plastid, is a process known as secondary endosymbiosis and is shown in a diverse group of eukaryotic organisms. Important members are heterokontophytes, haptophytes, cryptophytes, and apicomplexan parasites, all of them with complex plastids of red algal origin surrounded by four membranes. Although the evolutionary relationship between these organisms is still debated, they share common mechanisms for plastid protein import. In this review, we describe recent findings and current models on preprotein import into complex plastids with a special focus on the second outermost plastid membrane. Derived from the plasma membrane of the former endosymbiont, the evolution of protein transport across this so-called periplastidal membrane most likely represented the challenge in the transition from an endosymbiont to a host-dependent organelle. Here, remodeling and relocation of the symbiont endoplasmic reticulum-associated degradation (ERAD) machinery gave rise to a translocon complex termed symbiont-specific ERAD-like machinery and provides a fascinating insight into complex cellular evolution.     

Genome-based reconstruction of the protein import machinery in the secondary plastid of a chlorarachniophyte alga

Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.     

Making new out of old: recycling and modification of an ancient protein translocation system during eukaryotic evolution. Mechanistic comparison and phylogenetic analysis of ERAD, SELMA and the peroxisomal importomer

At first glance the three eukaryotic protein translocation machineries--the ER-associated degradation (ERAD) transport apparatus of the endoplasmic reticulum, the peroxisomal importomer and SELMA, the pre-protein translocator of complex plastids--appear quite different. However, mechanistic comparisons and phylogenetic analyses presented here suggest that all three translocation machineries share a common ancestral origin, which highlights the recycling of pre-existing components as an effective evolutionary driving force. Editor's suggested further reading in BioEssays ERAD ubiquitin ligases Abstract.     

Membrane heredity and early chloroplast evolution

Membrane heredity was central to the unique symbiogenetic origin from cyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae, glaucophytes) and to subsequent lateral transfers of plastids to form even more complex photosynthetic chimeras. Each symbiogenesis integrated disparate genomes and several radically different genetic membranes into a more complex cell. The common ancestor of Plantae evolved transit machinery for plastid protein import. In later secondary symbiogeneses, signal sequences were added to target proteins across host perialgal membranes: independently into green algal plastids (euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists). Conservatism and innovation during early plastid diversification are discussed.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Der1-mediated preprotein import into the periplastid compartment of chromalveolates?

Phototrophic chromalveolates possess plastids surrounded by either 3 or 4 membranes, revealing their secondary endosymbiotic origin from an engulfed eukaryotic alga. In cryptophytes, a member of the chromalveolates, the organelle is embedded within a designated region of the host's rough endoplasmic reticulum (RER). Its eukaryotic compartments other than the plastid were reduced to the mere remains of its former cytosol, the periplastid compartment (PPC, PP space), and its nucleus, the nucleomorph, separated from the RER by its former plasma membrane, the periplast membrane (PPM). In the nucleomorph genome of the cryptophyte Guillardia theta, we identified several genes sharing homology with components of the ER-associated degradation (ERAD) machinery of yeast and higher eukaryotes, namely ORF201 and ORF477, homologs of membrane-bound proteins, Der1p (Degradation in the ER protein 1) and the RING-finger ubiquitin ligase Hrd1, and a truncated version of Udf1, a cofactor of Cdc48, a lumenal ATPase. Exemplarily, studies on the Der1-homolog ORF201 showed that this protein partially rescued a yeast deletion mutant, indicating the existence of a functional PPC-specific ERAD-like system in cryptophytes. With the noninvestigated exception of haptophytes a phylogenetically and mechanistically related system is apparently present in all chromalveolates with 4 membrane-bound plastids because amongst others, PPC-specific Derlins (Der1-like proteins), CDC48 and its cofactor Ufd1 were identified in the nuclear genomes of diatoms and apicomplexa. These proteins are equipped with the required topogenic signals to direct them into the periplastid compartment of their secondary symbionts. Based on our findings, we suggest that all chromalveolates with 4 membrane-bound plastids express an ERAD-derived machinery in the PPM of their secondary plastid, coexisting physically and systematically adjacent to the host's own ERAD system. We propose herewith that this system was functionally adapted to mediate transport of nucleus-encoded PPC/plastid preproteins from the RER into the periplastid space.     

Protein Targeting into Secondary Plastids1

ABSTRACT. Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus‐encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus‐encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre‐existing ones to new requirements in secondary plastids.     

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast of Plasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.The apicomplexan parasite Plasmodium falciparum is the etiological agent of malaria tropica, the most severe form of human malaria, responsible for over 250 million infections and 1 million deaths annually (61). Many apicomplexan parasites, including P. falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event (27, 58). Although during the course of evolution this plastid organelle has lost the ability to carry out photosynthesis, it is still the site of several important biochemical pathways, including isoprenoid and heme biosynthesis, and as such is essential for parasite survival (60). As in other plastids, the vast majority of genes originally encoded on the plastid genome have been transferred to the nucleus of the host. As a result, their gene products (predicted to constitute up to 10% of all nucleus-encoded proteins) must be imported back into the apicoplast (12). The apicoplast is surrounded by four membranes (55), and this protein import process thus represents a major cell biological challenge and has attracted much research interest, not least due to the importance of P. falciparum as a human pathogen (16, 50).The signals directing transport of nucleus-encoded proteins to complex plastids, including the apicomplexan apicoplast, have been studied in great detail in recent years, and reveal that such proteins are endowed with specific N-terminal targeting sequences, referred to as a bipartite topogenic signals (BTS), that direct their transport to this compartment (50). BTS are composed of an N-terminal endoplasmic reticulum (ER)-type signal sequence, which initially allows proteins to enter the secretory system via the Sec61 complex (59). Following this, proteins are carried via a Golgi complex-independent transport step to the second outermost membrane, from where they are then translocated across the remaining three apicoplast membranes, directed by the second part of the BTS, the transit peptide (51). Based on evolutionary considerations, it has long been suggested that transport across the inner two apicoplast membranes occurs via a Toc/Tic-like (where Toc and Tic are translocons of the outer and inner chloroplast envelopes, respectively) protein translocase machinery, and this is supported by a recent publication that provides evidence for an essential role of a Toxoplasma gondii Tic20 homologue in this transport process (50, 57). Despite this progress, it is still unclear how proteins travel across the second and third outer apicoplast membranes. Several models have been discussed to account for this transport step, including vesicular shuttle and translocon-based mechanisms (recently reviewed in reference 19), but until recently no actual molecular equipment had been found which could account for these membrane translocation events. To address this question, Sommer et al. screened the nucleomorph genome of the chromalveolate cryptophyte Guillardia theta (which, similar to P. falciparum, contains a four-membrane-bound plastid organelle) for genes encoding potential translocon-related proteins (49). Surprisingly, the authors identified genes encoding proteins usually involved in the ER-associated protein degradation pathway (ERAD), which recognizes incorrectly folded protein substrates and retrotranslocates them to the cell cytosol for degradation by the ubiquitin (Ub)-proteasome system (35, 44). As such, the ERAD system functions as a translocation complex, capable of transporting proteins across a biological membrane. Further characterization of one of these proteins (G. theta Der1-1, a homologue of yeast Der1p, a component of the ERAD system) provided strong evidence for a plastid localization. These data suggested an attractive solution to the mechanistic problem of transport across the second and third outermost membrane of complex plastids by hypothesizing a role for an ERAD-derived protein translocon complex. Intriguingly, this study also identified several members of this ERAD-derived translocon complex (apicoplast ERAD [apERAD]) in the nuclear genome of P. falciparum endowed with an N-terminal BTS (49). The BTS derived from one of these proteins, P. falciparum sDer1-1 [PfsDer1-1], was sufficient to direct transport of green fluorescent protein (GFP) to the apicoplast of P. falciparum, suggesting that this ERAD-like machinery is ubiquitous among chromalveolates with four membrane-bound plastids (49). In this current report we extend our study of the P. falciparum apERAD complex.     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

The epoxy‐xanthophylls antheraxanthin and violaxanthin are key precursors of light‐harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP‐deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.     

From nuclear genes to chloroplast localized proteins

There is broad evidence that an endosymbiotic uptake of a cyanobacterial-type organism was the point of origin for the evolution of chloroplasts. During organelle evolution extensive gene transfer from the symbiont to the host genome occurred, which raises the question of how these gene products, namely proteins, which are still functional in chloroplasts, find their way back ‘home’. Nuclear-encoded proteins enter plastids via a complex import machinery that requires the coordinate interplay of a variety of soluble and membrane-bound factors on the cytosolic site as well as on the stromal side of the chloroplast envelope membranes. We define that the process called ‘import of chloroplast precursor proteins’ begins with the release of the polypeptide from the ribosomes and binding to cytosolic factors, such as a guidance complex, which accompanies (chaperones) proteins to chloroplasts. The translocation across the envelope membranes engages distinct translocation machineries at the outer and the inner envelope membranes. Additionally subsequent sorting events to different subcompartments within the plastids are operated by a number of distinct pathways, all of which seem to involve multiple subunits, which are largely of bacterial (symbiotic) origin. The evolutionary history of proteins mediating the import of chloroplast constituents across the envelope membranes seems more diverse. Since cyanobacteria lack a protein import pathway, it is not surprising that only a few subunits of the chloroplast translocon seem to be of symbiotic origin while others seem to be eukaryotic additions.     

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.     

Translocation of proteins across the multiple membranes of complex plastids.

Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.     

Evolution of Protein Targeting into “Complex” Plastids: The “Secretory Transport Hypothesis”

Abstract: In algae different types of plastids are known, which vary in pigment content and ultrastructure, providing an opportunity to study their evolutionary origin. One interesting feature is the number of envelope membranes surrounding the plastids. Red algae, green algae and glaucophytes have plastids with two membranes. They are thought to originate from a primary endocytobiosis event, a process in which a prokaryotic cyanobacterium was engulfed by a eukaryotic host cell and transformed into a plastid. Several other algal groups, like euglenophytes and heterokont algae (diatoms, brown algae, etc.), have plastids with three or four surrounding membranes, respectively, probably reflecting the evolution of these organisms by so‐called secondary endocytobiosis, which is the uptake of a eukaryotic alga by a eukaryotic host cell. A prerequisite for the successful establishment of primary or secondary endocytobiosis must be the development of suitable protein targeting machineries to allow the transport of nucleus‐encoded plastid proteins across the various plastid envelope membranes. Here, we discuss the possible evolution of such protein transport systems. We propose that the secretory system of the respective host cell might have been the essential tool to establish protein transport into primary as well as into secondary plastids.     

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.     

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.     

Endoplasmic reticulum-associated degradation of ricin A chain has unique and plant-specific features

Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.