Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

Okawa, Kumiko; Inoue, Hitoshi; Adachi, Fumi; Nakayama, Katsuhiro; Ito‐Inaba, Yasuko; Schnell, Danny J.; Uehara, Susumu; Inaba, Tomoki

doi:10.1093/jxb/eru290

Cited by 24 publications

(29 citation statements)

References 36 publications

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“…1). According to our previous study 12 , the topology of K124 has been shown to be flipped at the IEM, and the C-terminus faces toward the stroma. Hence, we assumed that the topology of a bicarbonate transporter fused to K124, SbtAIII, is reverted, as compared to that fused to the full-length Cor413im1, SbtAII 10 .…”

Section: Co-expression Of Chimeric Bicarbonate Transporters and Tev Pmentioning

confidence: 77%

“…The antibodies against LSU, Tic110, and Toc75 have been previously described 10,12,28,[31][32][33] . The LHCP antibodies were a kind gift from Prof. Kenneth Cline.…”

Section: Analysis Of the Localization Of Each Chimeric Protein Withinmentioning

confidence: 99%

“…Although we sometimes loaded a different amount of protein in the analysis, the protein ratio of total chloroplast to stroma to envelope to thylakoid was consistently 3:3:1:1.5. The trypsin sensitivity of the chimeric BicA and SbtA proteins was examined using intact chloroplasts as described previously [28][29][30] .The antibodies against LSU, Tic110, and Toc75 have been previously described 10,12,28,[31][32][33] . The LHCP antibodies were a kind gift from Prof. Kenneth Cline.…”

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confidence: 99%

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Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Uehara

Sei

Sada

et al. 2020

Sci Rep

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to improve the photosynthetic performance of c 3 plants, installing cyanobacterial bicarbonate transporters to the chloroplast inner envelope membrane (IEM) has been proposed for years. In our previous study, we successfully introduced chimeric cyanobacterial sodium-dependent bicarbonate transporters, BicA or SbtA, to the chloroplast IEM of Arabidopsis. However, the installation of authentic BicA and SbtA to the chloroplast IEM has not been achieved yet. In this study, we examined whether or not tobacco etch virus (TEV) protease targeted within chloroplasts can cleave chimeric proteins and produce authentic bicarbonate transporters. To this end, we constructed a TEV protease that carried the transit peptide and expressed it with chimeric BicA or SbtA proteins containing a TEV cleavage site in planta. Chimeric proteins were cleaved only when the TEV protease was co-expressed. The authentic forms of hemagglutinin-tagged BicA and SbtA were detected in the chloroplast IEM. In addition, cleavage of chimeric proteins at the teV recognition site seemed to occur after the targeting of chimeric proteins to the chloroplast IEM. We conclude that the cleavage of chimeric proteins within chloroplasts is an efficient way to install authentic bicarbonate transporters to the chloroplast IEM. Furthermore, a similar approach can be applied to other bacterial plasma membrane proteins.Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the incorporation of CO 2 into ribulose 1,5-bisphosphate (RuBP), which is indispensable for carbohydrate production in plants. However, Rubisco also catalyzes the oxygenation reaction of RuBP. This reaction has been considered wasteful since extra energy is consumed to recover RuBP and CO 2 is partially lost during the process of photorespiration. To compensate for the promiscuous nature of Rubisco, photosynthetic organisms have evolved various CO 2 -concentrating mechanisms (CCMs) 1,2 . For instance, cyanobacterial CCMs possess inorganic carbon (Ci) uptake systems and the microcompartments, carboxysomes, containing Rubisco. To date, five types of active Ci uptake systems have been identified in cyanobacteria. BicA and SbtA are single subunit sodium-dependent bicarbonate transporters on the plasma membrane 3,4 . In contrast, BCT1 is an ATP-binding, cassette-type bicarbonate transporter. The multimeric BCT1 complex is composed of four different subunits 5 . It has been proposed that the installation of CCM to chloroplasts is a promising approach to improve photosynthesis in C 3 plants. According to a theoretical estimation, installing any one of the bicarbonate transporters, BicA, BCT1, or SbtA alone, to the chloroplast inner envelope membrane (IEM) may improve photosynthesis 6-8 .It has been shown that chimeric cyanobacterial bicarbonate transporters expressed in the nucleus can be targeted to the chloroplast IEM 9,10 . Rolland et al. used a membrane protein leader (MPL) sequence that was fused to the N-terminus of the bicarbonate transporter together with the transit peptide 9 , which...

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Section: Co-expression Of Chimeric Bicarbonate Transporters and Tev Pmentioning

confidence: 77%

“…The antibodies against LSU, Tic110, and Toc75 have been previously described 10,12,28,[31][32][33] . The LHCP antibodies were a kind gift from Prof. Kenneth Cline.…”

Section: Analysis Of the Localization Of Each Chimeric Protein Withinmentioning

confidence: 99%

mentioning

confidence: 99%

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Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Uehara

Sei

Sada

et al. 2020

Sci Rep

Self Cite

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“…Instead, they may be subject to lateral insertion into the envelope membranes. Specific mitochondrial and chloroplast proteins are proposed to be inserted into the outer or inner envelope membrane by lateral translocation from the import channel (Viana et al, 2010;Okawa et al, 2014;Harner et al, 2011), and endoplasmic reticulum membrane proteins are laterally translocated from import channels to membranes (Ismail et al, 2012). The lateral translocation of inefficiently translocating preproteins with TMDs to outer envelope membranes may constitute a mechanism by which the import channel is preserved for efficient translocation of cargoes.…”

Section: Discussionmentioning

confidence: 99%

“…It is not fully understood how the pulling process occurs in chloroplasts, or whether TPs contain any sequence motifs that direct the pulling. Chloroplasts import soluble and membrane proteins that contain transmembrane domains (TMDs; Okawa et al, 2014;Viana et al, 2010;Froehlich and Keegstra, 2011;Lee et al, 2017). The mechanistic function of chloroplast import channels is not fully understood, and it is possible that specific properties of the polypeptides that pass through the import channels may affect their translocation.…”

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confidence: 99%

Prolines in Transit Peptides Are Crucial for Efficient Preprotein Translocation into Chloroplasts

et al. 2017

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Chloroplasts import many preproteins that can be classified based on their physicochemical properties. The cleavable N-terminal transit peptide (TP) of chloroplast preproteins contains all the information required for import into chloroplasts through Toc/Tic translocons. The question of whether and how the physicochemical properties of preproteins affect TP-mediated import into chloroplasts has not been elucidated. Here, we present evidence that Pro residues in TP mediate efficient translocation through the chloroplast envelope membranes for proteins containing transmembrane domains (TMDs) or proteins prone to aggregation. By contrast, the translocation of soluble proteins through the chloroplast envelope membranes is less dependent on TP prolines. Proless TPs failed to mediate protein translocation into chloroplasts; instead, these mutant TPs led to protein mistargeting to the chloroplast envelope membranes or nonspecific protein aggregation during import into chloroplasts. The mistargeting of TMD-containing proteins caused by Pro-less TPs in wild-type protoplasts was mimicked by wild-type TPs in protoplasts, in which preprotein translocation is compromised. We propose that the physicochemical properties of chloroplast proteins affect protein translocation through the chloroplast envelope, and prolines in TP have a crucial role in the efficient translocation of TMD-containing proteins.

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The N-region sequence context impacts the chloroplast import efficiency of multi-TMD protein

Nayak,

Mehrotra,

Mehrotra

2024

Plant Mol Biol

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Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

Abstract: Highlight textThis work demonstrated that the targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments including the indispensable transmembrane segment.

Cited by 24 publications

References 36 publications

Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Prolines in Transit Peptides Are Crucial for Efficient Preprotein Translocation into Chloroplasts

The N-region sequence context impacts the chloroplast import efficiency of multi-TMD protein

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