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The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum cha- perone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

Summary Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR‐Cas9 target‐specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse‐chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function., Significance Statement LPA2 is a nuclear‐encoded protein that is suggested to be involved in PSII assembly in vascular plants. Here, using CRISPSRS/CAS9 mutagenesis, we identified and investigated an LPA2 homolog in Chlamydomonas reinhardtii. lpa2 mutants were characterized by a strong reduction in the accumulation of photosystem II core subunits, reduced photosystem II activity, increased photosensitivity and impairment in photoautotrophic growth, revealing the critical role of LPA2 in the assembly of photosystem II supercomplexes in green algae.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

Plastids are sites of biochemical and biological processes that are fundamental for plant life. The genome of these endosymbiotic organelles encodes for almost one hundred of the three thousand proteins that make up the chloroplast proteome. Genes coding for plastid multi-subunit protein complexes derive from both nuclear and plastid genomes, so it is clear that there is the need of a highly integrated coordination between this two subcellular compartments. The coordination between the nucleus and the plastome takes place at many different levels, including the modulation of nuclear and plastid transcription, RNA processing and translation, post-translational modifications and protein targeting. In addition, the plastid retains a whole series of mechanisms for the preservation of its protein balance (proteostasis), including proteases and molecular chaperones (Figure 1). Plastids have largely abandoned transcriptional control switching predominantly to translational and post-translational control of their gene expression, but some transcriptional regulation is known to occur. Transcription of plastid genes is performed by two different types of RNA polymerases: plastid-encoded RNA polymerase (PEP) and a nuclear-encoded RNA polymerases (NEP). Liebers et al. (2017) propose that targeted changes in plastid transcription, mostly by controlling the relative activities of NEP and PEP enzymes, impact the establishment of the plastid proteome and these represent key determinants for the transitions between the different plastid types. The transcriptional regulation mechanisms are still far from being completely elucidated. An unusual light- and stress-responsive promoter (psbD LRP), regulated by a AAG-box immediately upstream of the -35 element, has been recently mapped. Shinmura et al. (2017) analysed psbD LRP promoter regions in eleven embryophytes, at different evolutionary stages, from liverworts to angiosperms. This analysis identified conserved features of the promoter and facilitated study of its emergence and evolution in plant species. Among the proteins that regulate plastid gene expression, the nucleus-encoded proteins of the mitochondrial transcription termination factor (mTERF) family have been recently identified. Information on their function is only beginning to emerge. Xu et al. (2017) investigate the function of the chloroplast-associated mTERF. They report that these proteins are localized to chloroplast nucleoids and identify two of them involved in the salt stress response. The import of plastid precursor proteins into plastids is another checkpoint affecting plastid proteostasis that is regulated in response to the fluctuating environmental conditions. This fine regulation ensures the optimal functioning of important biological processes taking place in this cellular compartment. The import of plastid precursor proteins is mediated by two distinct translocation complexes called TOC and TIC, located respectively at the outer and at the inner envelope membrane of chloroplasts. The individual steps involved in protein translocation and the corresponding regulation mechanisms used by plants to modulate protein import are reviewed by Sjuts et al. (2017). Upon transition from an endosymbiont to a plant cell organelle, the plastid retains a set of mechanisms that involves enzymes and proteins of prokaryotic origin which are responsible for protein maturation, post-translational modification, correct folding, protein abundance control and removal of misfolded or damaged components. These processes ensure that plastid proteins are ready to exert their biological mission and require an intricate system of signals from nucleus to plastid and backwards. Plastid-derived signals can regulate availability of nuclear-encoded plastid precursors controlling their de novo synthesis, and targeting. Recently, an important player in the chloroplast-to-nucleus retrograde communication has been identified: the protein GENOMES UNCOUPLED1 (GUN1). Recent studies indicate that GUN1 might play a role in the coordination of translation, import and degradation of plastid proteins. The molecular function of different GUN1 partners has been reviewed by Colombo et al. (2016), highlighting its potential role in plastid proteostasis. Another important mechanism during nuclear-plastid interaction is the degradation of multiple components through the ubiquitin-proteasome system. It has become increasingly clear that, together with feedback regulation of nuclear gene expression by plastid-derived signals, this mechanism avoids the accumulation of non-imported proteins in the cytosol. In addition to the anterograde signaling pathway, recent studies in A. thaliana demonstrated that also the retrograde signaling pathway can be subjected to ubiquitin-proteasome regulation. Hirosawa et al. (2017) review recent advances in understanding how the ubiquitin-proteasome system regulates the nuclear-plastid interaction and plastid biogenesis. The maturation of plastid proteins is another highly regulated process that in some cases needs complex apparatuses to occur. This is the case of c-type cytochromes that require a multicomponent assembly pathway for their maturation, as reviewed by Gabilly et al. (2017). The regulation of this pathway has not yet been clarified. Cline et al. (2017) describe the functional characterization of the CCS2 gene of Chlamydomonas reinhardtii required for cytochrome c assembly. The authors discuss the possible functions of CCS2 in the heme attachment reaction. A wide range of post-translational modifications (PTMs) contribute to finely regulate the biological processes that take place in plastids. PTMs alter the physicochemical properties of the plastidial proteins thus affecting their function. Grabsztunowicz et al. (2017) review the current knowledge on the PTMs regulating important metabolic processes in chloroplasts such as DNA replication and gene expression, photosynthetic carbon assimilation and starch metabolism and report the known physiological effects of these modifications. Considering that there is limited knowledge of the combined action among the molecular mechanisms that regulate plastid proteostasis, the goal of this Research Topic is to bring together a set of articles that contribute to our understanding of how transcription, translation, and post-translational modifications (including protein targeting) maintain plastid proteostasis. As a consequence, this Research Topic will help us understand more deeply how plastids function.

The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum.

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on "Unconventional Protein and Membrane Traffic" (UPMT) during 4-7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast.

The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence.

Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd. However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues.

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Studies on the basic mechanisms that regulate vacuolar delivering of proteins synthesized in the endoplasmic reticulum (ER) have a great importance in plant cell biology. Indeed, many aspects of plant physiology are affected by this intracellular traffic, for example, germination or reaction to biotic stresses due to the accumulation of storage proteins in seeds or enzymes in vegetative tissues, respectively. Up to now, the Golgi complex has been considered the main hub in the sorting of vacuolar secretory proteins; those polypeptides able to reach their final destination without the aid of this organelle are regarded as exceptions to an established route. This mini-review aims to emphasize the existence of several Golgi-independent pathways involved in the trafficking of different types of vacuolar proteins.

The insertion of transgenes into the plastid genome (plastome) has proved to be an effective alternative to nuclear transformation for producing stable transformants expressing heterologous proteins. Recently, methodology, characteristics and possible fields of application of plastome transformation have been extensively reviewed (Lössl and Waheed, 2011; Scotti et al., 2012). From these reports, plastid transformation turns out to be a very promising tool for the production of recombinant proteins in plants, yet some limitations must still be overcome. Although the plastome of many species has been successfully transformed, an efficient and reproducible transformation procedure easily adoptable by any plant laboratory has been established only in tobacco, and to a minor extent in two other solanaceous species (potato and tomato), as well as in few other species (Maliga and Bock, 2011). Moreover, up to now the steady-state level of a recombinant protein coded by a gene inserted into the plastome has failed to be predictable. In spite of a huge number of reports on successful foreign protein production in plastids, above all proteins with a pharmaceutical interest, in many cases the expression level of other recombinant proteins in the transplastomic plants appears to be very low or even undetectable (e.g. Birch-Machin et al., 2004; Bellucci et al., 2005; Wirth et al., 2006). We believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of protein homeostasis, or proteostasis. This term refers to a complex network of biological pathways capable of maintaining the dynamic equilibrium of the protein pool, thus allowing the cell to get used to environmental changes (Balch et al., 2008). This concept can also be extended to subcellular level, especially for those organelles with an endosymbiotic origin like the plastids and mitochondria (Waller, 2012). These organelles retain a whole series of mechanisms for the preservation of their protein balance, including specific proteases, transcriptional and translational control, as well as molecular chaperones and enzymes useful in protein folding. Therefore, it is important to develop basic studies in factors regulating protein synthesis, stability, folding, targeting, and accumulation in plastids, including the protein quality control which contributes to the functional integrity of proteins. Our intention here is not to provide a comprehensive review of the mechanisms that regulate plastid proteostasis, but to discuss some recent insights into this field which might bring beneficial applications in plastid biotechnology for transgene expression and foreign protein accumulation.

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin γ-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like γ-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.