Dual Protein Localization to the Envelope and Thylakoid Membranes Within the Chloroplast

Klasek, Laura; Inoue, Kentaro

doi:10.1016/bs.ircmb.2015.12.008

Cited by 11 publications

(12 citation statements)

References 141 publications

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“…Experiments using increasing external ATP concentrations indicate that TROL requires more than 300 mM ATP for completion of its import into thylakoids, while only 30 mM is necessary for IM incorporation, as visible after thermolysin treatment ( Figure 3 ). This result implies the stop-transfer mechanism of TROL import and its lateral insertion to the IM [ 11 , 22 ]. Time-scale experiments in the presence of 3 mM ATP indicate that the incorporation of TROL into the IM happens very early, and the smaller thylakoid form starts to appear after 2–5 min of import, reaching a maximum between 10–20 min ( Figure 4 ).…”

Section: Discussionmentioning

confidence: 99%

“…TROL is not an isolated case in terms of dual localization in chloroplasts. Seventeen other proteins have been identified in both chloroplastic envelope and thylakoid membranes [ 22 ]. All of them are predicted to carry an N-terminal signal for chloroplast import.…”

Section: Discussionmentioning

confidence: 99%

“…Further import into thylakoids requires additional translocators and energy demand. Four different mechanisms are known to target proteins from the stroma to the thylakoids [ 20 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

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Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Vojta

Čuletić

Fulgosi

2018

IJMS

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Thylakoid rhodanase-like protein (TROL) is involved in the final step of photosynthetic electron transport from ferredoxin to ferredoxin: NADP+ oxidoreductase (FNR). TROL is located in two distinct chloroplast compartments—in the inner envelope of chloroplasts, in its precursor form; and in the thylakoid membranes, in its fully processed form. Its role in the inner envelope, as well as the determinants for its differential localization, have not been resolved yet. In this work we created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain a construct whose import is affected or localization limited to a single intrachloroplastic site. By using in vitro transcription and translation and subsequent protein import methods, we found that a single amino acid exchange in the presequence, Ala67 to Ile67 interferes with processing in the stroma and directs the whole pool of in vitro translated TROL to the inner envelope of chloroplasts. This result opens up the possibility of studying the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Vojta

Čuletić

Fulgosi

2018

IJMS

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“…Biochemical experiments in vitro demonstrated that Plsp1 is inserted into the thylakoid membrane through two independent pathways: (1) SEC, which requires the transmembrane domain (TMD) of Plsp1 owing to the lack of cleavable TTSs, and (2) the spontaneous pathway, which is prevented by sequestering Plsp1 into a stromal 700-kDa complex, which likely contains chaperonin-60, due to the high frequency of Plsp1 missorting (Endow et al, 2015). Plsp1 is also anchored to the inner envelope and its proteolytic domain is presumably exposed to the intermembrane space (Shipman and Inoue, 2009;Klasek and Inoue, 2016). While this configuration needs experimental verification, it well explains its role in Toc75 protein processing (Inoue et al, 2005).…”

Section: Prep1/2mentioning

confidence: 99%

Essentials of Proteolytic Machineries in Chloroplasts

2017

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Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic processes, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by the balanced protein synthesis and degradation, which is depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remodeling, and maintenance. Recently identified putative proteases are potential regulators of photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation machineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides.

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“…n75 acts as a canonical transit peptide and is removed in the stroma [34, 35]. c75 is necessary but not sufficient for targeting Toc75 to the OEM [35] and is removed by membrane-bound plastidic type I signal peptidase 1 (Plsp1) most likely at the intermembrane space (IMS) [36–39]. Within c75 are two conserved regions, a hydrophobic domain (residues 52–77 in psToc75) and a region containing polyGly (residues 91–110 in psToc75) [3] (Fig 1A).…”

Section: Introductionmentioning

confidence: 99%

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

et al. 2016

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PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

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Dual Protein Localization to the Envelope and Thylakoid Membranes Within the Chloroplast

Cited by 11 publications

References 141 publications

Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Essentials of Proteolytic Machineries in Chloroplasts

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

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