scholarly journals Sequence analysis and protein interactions of Arabidopsis CIA2 and CIL proteins

2020 ◽  
Author(s):  
Chun-Yen Yang ◽  
Chih-Wen Sun
Keyword(s):  
Protein Interactions ◽  
Protein Transport ◽  
Protein Import ◽  
Leaf Shape ◽  
Homologous Proteins ◽  
Protein Fragments ◽  
Nuclear Localization Signals ◽  
Flowering Control ◽  
Reduced Rate ◽  
Pale Green

Abstract Background: A previous screening of Arabidopsis thaliana for mutants exhibiting dysfunctional chloroplast protein transport identified the chloroplast import apparatus ( cia ) gene. The cia2 mutant has a pale green phenotype and reduced rate of protein import into chloroplasts, but leaf shape and size are similar to wild-type plants of the same developmental stage. Microarray analysis showed that nuclear CIA2 protein enhances expression of the Toc75 , Toc33 , CPN10 and cpRPs genes, thereby up-regulating protein import and synthesis efficiency in chloroplasts. CIA2-like (CIL) shares 65% sequence identity to CIA2, suggesting that CIL and CIA2 are homologous proteins in Arabidopsis. Here, we further assess the protein interactions and sequence features of CIA2 and CIL. Results: Subcellular localizations of truncated CIA2 protein fragments in our onion transient assay demonstrate that CIA2 contains two nuclear localization signals (NLS) located at amino acids (aa) 62-65 and 291-308, whereas CIL has only one NLS at aa 47-50. We screened a yeast two-hybrid (Y2H) Arabidopsis cDNA library to search for putative CIA2-interacting proteins and identified 12 nuclear proteins, including itself, CIL, and flowering-control proteins (such as CO, NF-YB1, NF-YC1, NF-YC9 and ABI3). Additional Y2H experiments demonstrate that CIA2 and CIL mainly interact with flowering-control proteins via their N-termini, but preferentially form homo- or hetero-dimers through their C-termini. Moreover, sequence alignment showed that the N-terminal sequences of CIA2, CIL and NF-YA are highly conserved. Therefore, NF-YA in the NF-Y complex could be substituted by CIA2 or CIL. Conclusions: We show that Arabidopsis CIA2 and CIL can interact with CO and NF-Y complex, so not only may they contribute to regulate chloroplast function but also to modulate flower development.

scholarly journals Sequence analysis and protein interactions of Arabidopsis CIA2 and CIL proteins

2020 ◽  
Author(s):  
Chih-Wen Sun ◽  
Chun-Yen Yang
Keyword(s):  
Protein Interactions ◽  
Protein Transport ◽  
Protein Import ◽  
Leaf Shape ◽  
Homologous Proteins ◽  
Protein Fragments ◽  
Nuclear Localization Signals ◽  
Flowering Control ◽  
Reduced Rate ◽  
Pale Green

Abstract Background: A previous screening of Arabidopsis thaliana for mutants exhibiting dysfunctional chloroplast protein transport identified the chloroplast import apparatus ( cia ) gene. The cia2 mutant has a pale green phenotype and reduced rate of protein import into chloroplasts, but leaf shape and size are similar to wild-type plants of the same developmental stage. Microarray analysis showed that nuclear CIA2 protein enhances expression of the Toc75 , Toc33 , CPN10 and cpRPs genes, thereby up-regulating protein import and synthesis efficiency in chloroplasts. CIA2-like (CIL) shares 65% sequence identity to CIA2, suggesting that CIL and CIA2 are homologous proteins in Arabidopsis. Here, we further assess the protein interactions and sequence features of CIA2 and CIL. Results: Subcellular localizations of truncated CIA2 protein fragments in our onion transient assay demonstrate that CIA2 contains two nuclear localization signals (NLS) located at amino acids (aa) 62-65 and 291-308, whereas CIL has only one NLS at aa 47-50. We screened a yeast two-hybrid (Y2H) Arabidopsis cDNA library to search for putative CIA2-interacting proteins and identified 12 nuclear proteins, including itself, CIL, and flowering-control proteins (such as CO, NF-YB1, NF-YC1, NF-YC9 and ABI3). Additional Y2H experiments demonstrate that CIA2 and CIL mainly interact with flowering-control proteins via their N-termini, but preferentially form homo- or hetero-dimers through their C-termini. Moreover, sequence alignment showed that the N-terminal sequences of CIA2, CIL and NF-YA are highly conserved. Therefore, NF-YA in the NF-Y complex could be substituted by CIA2 or CIL. Conclusions: We show that Arabidopsis CIA2 and CIL can interact with CO and NF-Y complex, so not only may they contribute to regulating chloroplast function but also to modulating flower development.

Regulation of protein transport to the nucleus: central role of phosphorylation

1996 ◽  
Vol 76 (3) ◽  
pp. 651-685 ◽  
Author(s):  
D. A. Jans ◽  
S. Hubner
Keyword(s):  
Nuclear Localization ◽  
Protein Transport ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Nuclear Accumulation ◽  
Control Of Gene Expression ◽  
Nuclear Localization Signals

Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression. Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated. Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast. Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport. Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation. The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function. The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacteriumColwellia psychrerythraea34H

2014 ◽  
Vol 70 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Hackwon Do ◽  
Chang Woo Lee ◽  
Se Jong Han ◽  
Sung Gu Lee ◽  
Hak Jun Kim ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Aromatic Acid ◽  
Diffusion Method ◽  
Flavin Mononucleotide ◽  
Acid Decarboxylase ◽  
Data Sets ◽  
Single Mutation ◽  
Homologous Proteins ◽  
Space Group

TheubiXgene (UniProtKB code Q489U8) ofColwellia psychrerythraeastrain 34H has been annotated as a putative flavin mononucleotide (FMN)-dependent aromatic acid decarboxylase. Based on previous studies of homologous proteins, CpsUbiX is thought to catalyze the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to produce 2-polyprenylphenol in the ubiquinone-biosynthesis pathway using a noncovalently bound FMN molecule as a cofactor. However, the detailed mechanisms of this important enzyme are not yet clear and need to be further elucidated. In this study, it was found that the V47S single mutation resulted in a loss of FMN binding, resulting in the production of FMN-free CpsUbiX protein. This mutation is likely to destabilize FMN–protein interactions without affecting the overall structural folding. To fully characterize the conformational changes upon FMN binding and the enzymatic mechanism at the molecular level, the wild-type (FMN-bound) and V47S mutant (FMN-free) CpsUbiX proteins were purified and crystallized using the sitting-drop vapour-diffusion method. Furthermore, complete diffraction data sets for FMN-bound (space groupC2221) and FMN-free (space groupP23) forms were obtained to 2.0 and 1.76 Å resolution, respectively.

scholarly journals Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis

2020 ◽  
Vol 76 (12) ◽  
pp. 1244-1255
Author(s):  
Sandra Kozak ◽  
Yehudi Bloch ◽  
Steven De Munck ◽  
Aleksandra Mikula ◽  
Isabel Bento ◽  
...  
Keyword(s):  
Cell Line ◽  
Protein Interactions ◽  
Successful Implementation ◽  
Structural Studies ◽  
Protein Fragments ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Acid Protein ◽  
Stimulating Factor

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.

scholarly journals A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

1991 ◽  
Vol 113 (6) ◽  
pp. 1243-1254 ◽  
Author(s):  
U Stochaj ◽  
M Osborne ◽  
T Kurihara ◽  
P Silver
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Protein Localization ◽  
Binding Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Localization Signals

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

scholarly journals Complete Genome Sequence and In Planta Subcellular Localization of Maize Fine Streak Virus Proteins

2005 ◽  
Vol 79 (9) ◽  
pp. 5304-5314 ◽  
Author(s):  
Chi-Wei Tsai ◽  
Margaret G. Redinbaugh ◽  
Kristen J. Willie ◽  
Sharon Reed ◽  
Michael Goodin ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Protein Interactions ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Streak Virus ◽  
In Planta ◽  
Nuclear Localization Signals ◽  
N Protein ◽  
P Protein ◽  
Maize Fine Streak Virus

ABSTRACT The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.

scholarly journals Control of protein trafficking by reversible masking of transport signals

2016 ◽  
Vol 27 (8) ◽  
pp. 1310-1319 ◽  
Author(s):  
Omer Abraham ◽  
Karnit Gotliv ◽  
Anna Parnis ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Protein Import ◽  
Binding Peptide ◽  
Protein Traffic ◽  
Alternative Approach ◽  
Targeting Signal ◽  
Regulated Trafficking ◽  
Streptavidin Binding

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

scholarly journals Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

1997 ◽  
Vol 136 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Mitsuru Akita ◽  
Erik Nielsen ◽  
Kenneth Keegstra
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
Protein Import ◽  
Cross Linking ◽  
Envelope Membrane ◽  
Precursor Proteins ◽  
Intact Chloroplasts ◽  
Outer Envelope Membrane ◽  
Envelope Membranes ◽  
Chemical Cross Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

scholarly journals The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Mingjiu Li ◽  
Hannes Ruwe ◽  
Michael Melzer ◽  
Astrid Junker ◽  
Goetz Hensel ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Ribosomal Proteins ◽  
Protein Import ◽  
Chloroplast Development ◽  
Transit Peptide ◽  
Growth Conditions ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Chloroplast Transit Peptide ◽  
Pale Green

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

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