•Chloroplasts have three membranes, outer/inner envelopes and thylakoid membranes.•AKR2 coordinates cytosolic sorting and insertion of proteins into outer envelope.•Two pathways operate for ...biogenesis of inner envelope proteins.•The cpSRP pathway plays a key role in biogenesis of LHCPs into thylakoid membranes.•Besides LHCPs, most thylakoid membrane proteins are inserted spontaneously.
Among the many organelles in eukaryotic cells, chloroplasts have the most complex structure, with multiple suborganellar membranes, making protein targeting to chloroplasts, particularly to various suborganellar membranes, highly challenging. Multiple mechanisms function in the biogenesis of chloroplast membrane proteins. Nuclear-encoded nascent proteins can be targeted to the outer envelope membrane directly from the cytosol after translation, but their targeting to the inner envelope and thylakoid membranes requires multiple steps, including cytosolic sorting, translocation across the envelope membranes, sorting in the stroma, and insertion into their target membranes. In this review, we discuss the current knowledge about the sorting mechanisms of proteins to the two envelope membranes and the thylakoid membrane, along with perspectives for future research.
Engineered nanomaterials (ENMs) enable the control and exploration of intermolecular interactions inside microscopic systems, but the potential environmental impacts of their inevitable release ...remain largely unknown. Plants exposed to ENMs display effects, such as increase in biomass and chlorophyll, distinct from those induced by exposure to their bulk counterparts, but few studies have addressed the mechanisms underlying such physiological results. The current investigation found that exposure of Arabidopsis thaliana to nano zerovalent iron (nZVI) triggered high plasma membrane H+-ATPase activity. The increase in activity caused a decrease in apoplastic pH, an increase in leaf area, and also wider stomatal aperture. Analysis of gene expression indicated that the levels of the H+-ATPase isoform responsible for stomatal opening, AHA2, were 5-fold higher in plants exposed to nZVI than in unexposed control plants. This is the first study to show that nZVI enhances stomatal opening by inducing the activation of plasma membrane H+-ATPase, leading to the possibility of increased CO2 uptake.
Abstract
Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in
E
.
coli
, it is inevitable that LPS contaminates. However, LPS ...removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1–3 domains (CES3) of Factor C from the horseshoe crab
Carcinoscorpius rotundicauda
to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in
Nicotiana benthamiana
and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing
E
.
coli
. To produce CES3:CBM3 in an LPS-free environment, we generated
Arabidopsis
transgenic plants harbouring a recombinant gene,
BiP:NT:SUMO:CES3:CBM3:HDEL
, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from
Arabidopsis
plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.
Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle‐specific proteins. The majority of chloroplast and ...mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N‐terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15th and 21st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31st to 40th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41st to 50th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull‐down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual‐targeting AtSufE1 is mediated by both mitochondria‐specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.
Mitochondrial targeting of dual‐targeting AtSufE1 is mediated by both mitochondria‐specific and common sequence motifs in the signal sequence through the interaction with two import receptors, Metaxin and Tom20, in a successive manner.
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.
Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. ...These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.
► Various cytosolic events are required for protein targeting into interior regions of chloroplasts. ► Unimported precursor response plays a crucial role in regulation of cytosolic levels of chloroplast preproteins. ► Various cytosolic events are required for targeting to the outer envelope of chloroplasts. ► AKR2 and Hsp17.8 function as a cytosolic factor and cofactor for targeting to chloroplast outer membrane.
Although the chloroplasts in plants are characterized by an inherent genome, the chloroplast proteome is composed of proteins encoded by not only the chloroplast genome but also the nuclear genome. ...Nuclear-encoded chloroplast proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the chloroplasts. In the latter process, an N-terminal cleavable transit peptide serves as a targeting signal required for the import of nuclear-encoded chloroplast interior proteins. This import process is mediated
via
an interaction between the sequence motifs in transit peptides and the components of the TOC/TIC (translocon at the outer/inner envelope of chloroplasts) translocons. Despite a considerable diversity in primary structures, several common features have been identified among transit peptides, including N-terminal moderate hydrophobicity, multiple proline residues dispersed throughout the transit peptide, preferential usage of basic residues over acidic residues, and an absence of N-terminal arginine residues. In this review, we will recapitulate and discuss recent progress in our current understanding of the functional organization of sequence elements commonly present in diverse transit peptides, which are essential for the multi-step import of chloroplast proteins.
ABSTRACT
Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, ...organellar proteins carry an organelle‐specific targeting signal for recognition by organelle‐specific import machinery. However, the situation is confusing for transmembrane domain (TMD)‐containing signal‐anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine‐rich region flanking the N‐ and C‐termini of TMDs, respectively, and an aromatic residue in the C‐terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co‐translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post‐translational manner.
Mitochondrial targeting of signal‐anchored proteins requires multiple motifs around and within the transmembrane domain (TMD) such as a basic residue and an arginine‐rich region flanking the N‐ and C‐termini of TMDs, respectively, and an aromatic residue on the C‐terminal side of the TMD that supports co‐translational targeting.
Infection with human papillomavirus (HPV) can cause cervical cancers in women, and vaccination against the virus is one of most effective ways to prevent these cancers. Two vaccines made of ...virus-like particles (VLPs) of HPV L1 proteins are currently commercially available. However, these HPV vaccines are highly expensive, and thus not affordable for women living in developing countries. Therefore, great demand exists to produce a cost-effective vaccine. Here, we investigate the production of self-assembled HPV16 VLPs in plants. We generated a chimeric protein composed of N-terminal 79 amino acid residues of RbcS as a long-transit peptide to target chloroplasts, the SUMO domain, and HPV16 L1 proteins. The chimeric gene was expressed in plants with chloroplast-targeted bdSENP1, a protein that specifically recognizes the SUMO domain and cleaves its cleavage site. This co-expression of bdSENP1 led to the release of HPV16 L1 from the chimeric proteins without any extra amino acid residues. HPV16 L1 purified by heparin chromatography formed VLPs that mimicked native virions. Moreover, the plant-produced HPV16 L1 VLPs elicited strong immune responses in mice without adjuvants. Thus, we demonstrated the cost-effective production of HPV16 VLPs in plants.
Subcellular organelles in eukaryotes are surrounded by lipid membranes. In an endomembrane system, vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific ...organelles. However, organellar proteins for chloroplasts, mitochondria, the nucleus, and peroxisomes that are translated in the cytosol are directly imported into their target organelles. Chloroplasts are a plant‐specific organelle with outer and inner envelope membranes, a dual‐membrane structure that is similar to mitochondria. Interior chloroplast proteins translated by cytosolic ribosomes are thus translocated through TOC and TIC complexes (translocons in the outer and inner envelope of chloroplasts, respectively), with stromal ATPase motor proteins playing a critical role in pulling pre‐proteins through these import channels. Over the last three decades, the identity and function of TOC/TIC components and stromal motor proteins have been actively investigated, which has shed light on the action mechanisms at a molecular level. However, there remains some disagreement over the exact composition of TIC complexes and genuine stromal motor proteins. In this review, we discuss recent findings on the mechanisms by which proteins are translocated through TOC/TIC complexes and discuss future prospects for this field of research.
This review discusses recent progress in understanding how nuclear‐encoded chloroplast proteins enter chloroplasts, with a focus on preprotein translocation across TOC (translocon at the outer envelope of chloroplasts) and TIC (translocon at the inner envelope of chloroplasts) complexes, in which stromal motor proteins play a crucial role.
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