Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts

Campbell, James H.; Hoang, Thu Trang; Jelokhani-Niaraki, Masoud; Smith, Matthew D.

doi:10.1186/s12858-014-0029-y

Cited by 7 publications

(6 citation statements)

References 48 publications

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“…The θ 208 /θ 222 ratios of the CD spectra, in both OG detergent and liposomes, were less than 1 (0.60, 0.63 and 0.61 for UCP2, UCP4 and UCP5 respectively). Such ellipticity ratios have been observed in the spectra of coiled coil and helical bundle motifs and as a result of protein association [22,31–34]. Using complementary electrophoresis and MS methods, the atypical helical conformation of the neuronal UCPs can mostly probably be attributed to a predominant population of UCP multimers (tetramers) in liposomes with molecular mass of ∼ 140–150 kDa (Figures 2G–2I; Supplementary Table S1).…”

Section: Resultsmentioning

confidence: 75%

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

et al. 2015

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SynopsisMitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins selfassociate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCPmediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.

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Section: Resultsmentioning

confidence: 75%

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

et al. 2015

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“…The Tic20 gene family encodes an inner chloroplast membrane protein that likely functions as a channel for protein import (Kasmati et al, 2011;Kikuchi et al, 2013;Campbell et al, 2014). The severe tic20-iv phenotype on spectinomycin indicates that ACC2 is unable to enter the chloroplast and contribute to fatty acid biosynthesis when Tic20-IV is eliminated.…”

Section: Loss Of Tic20-iv-mediated Chloroplast Protein Import Resultsmentioning

confidence: 99%

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation

2016

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Natural accessions of Arabidopsis (Arabidopsis thaliana) differ in their ability to tolerate a loss of chloroplast translation. These differences can be attributed in part to variation in a duplicated nuclear gene (ACC2) that targets homomeric acetyl-coenzyme A carboxylase (ACCase) to plastids. This functional redundancy allows limited fatty acid biosynthesis to occur in the absence of heteromeric ACCase, which is encoded in part by the plastid genome. In the presence of functional ACC2, tolerant alleles of several nuclear genes, not yet identified, enhance the growth of seedlings and embryos disrupted in chloroplast translation. ACC2 knockout mutants, by contrast, are hypersensitive. Here we describe an expanded search for hypersensitive accessions of Arabidopsis, evaluate whether all of these accessions are defective in ACC2, and characterize genotype-to-phenotype relationships for homomeric ACCase variants identified among 855 accessions with sequenced genomes. Null alleles with ACC2 nonsense mutations, frameshift mutations, small deletions, genomic rearrangements, and defects in RNA splicing are included among the most sensitive accessions examined. By contrast, most missense mutations affecting highly conserved residues failed to eliminate ACC2 function. Several accessions were identified where sensitivity could not be attributed to a defect in either ACC2 or Tic20-IV, the chloroplast membrane channel required for ACC2 uptake. Overall, these results underscore the central role of ACC2 in mediating Arabidopsis response to a loss of chloroplast translation, highlight future applications of this system to analyzing chloroplast protein import, and provide valuable insights into the mutational landscape of an important metabolic enzyme that is highly conserved throughout eukaryotes.

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“…Earlier studies performed on pea chloroplasts revealed several of its subunits, including Tic110, Tic62, Tic55, Tic40, Tic32, Tic22, and Tic20 (21,(24)(25)(26)(27)(28). However, there is no consensus as to which components form the proteinconducting channel: while multiple studies demonstrated a channel activity for Tic20 in vitro (42)(43)(44)(45), it is still debated whether Tic110 is also directly involved in forming a channel (46)(47)(48)(49). The scenario has been further complicated by the recent isolation of a novel large TIC complex in Arabidopsis, termed the "1-MDa complex" (42,44), that contains Tic20 in association with a different set of proteins, namely Tic100, Tic56 and Tic214.…”

Section: Introductionmentioning

confidence: 99%

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

Ramundo

Asakura

eacute

et al. 2020

Preprint

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AbstractIn photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons — termed TOC and TIC — located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56 and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TicToc), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and the here newly identified TicToc components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TicToc complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TicToc supercomplex in maintaining chloroplast proteostasis.

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Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts

Cited by 7 publications

References 48 publications

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

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