Mitochondrial and chloroplastic targeting signals of NADP+-dependent isocitrate dehydrogenase

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (Nicotiana tabacum L.), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, suggesting the possibility of tandem targeting signals. In this work, the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) reporter to generate a series of constructs and introduced into tobacco leaves by Agrobacterium-mediated transient transformation. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined using confocal microscopy. Constructs predicted to be targeted to the chloroplast all localized to the chloroplast. However, this was not the case for all of the constructs that were predicted to be mitochondrial targeted. Although some constructs localized to mitochondria as expected, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein that were present in those constructs, generated from either ‘Xanthi’ or ‘Petit Havana’ cultivars of tobacco. The results of this study raise interesting questions regarding the targeting and processing of organellar isoforms of NADP+-ICDH.

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Targeting and processing of membrane-anchored YFP fusion proteins to protein storage vacuoles in transgenic tobacco seeds

Seed Science Research ◽

10.1079/ssr2005225 ◽

2005 ◽

Vol 15 (4) ◽

pp. 361-364 ◽

Cited By ~ 4

Author(s):

Ka Leung Fung ◽

Yin Fun Yim ◽

Yu Chung Tse ◽

Yansong Miao ◽

Samuel S.M. Sun ◽

...

Keyword(s):

Transgenic Tobacco ◽

Fusion Proteins ◽

Seed Protein ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Pharmaceutical Proteins ◽

Tobacco Seeds ◽

Foreign Proteins

Seeds that store proteins in protein storage vacuoles are attractive bioreactors for producing and storing large amounts of pharmaceutical proteins. However, foreign proteins expressed in transgenic plants are subjected to the delivery and modification processes present within plant cells. Here, it is demonstrated that unique membrane sequences deliver a yellow fluorescent protein (YFP) to the seed protein storage vacuoles in transgenic tobacco (Nicotiana tabacum L.) plants, where the YFP is then separated from its membrane anchors. This precise targeting and separation is required for the successful delivery of useful proteins to seed protein storage vacuoles for their stable accumulation in transgenic crops.

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Identification of sequences required for the import of human protoporphyrinogen oxidase to mitochondria

Biochemical Journal ◽

10.1042/bj20030978 ◽

2004 ◽

Vol 377 (2) ◽

pp. 281-287 ◽

Cited By ~ 10

Author(s):

Rhian R. MORGAN ◽

Rachel ERRINGTON ◽

George H. ELDER

Keyword(s):

Mitochondrial Membrane ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Missense Mutations ◽

Amino Acid Residues ◽

Inner Mitochondrial Membrane ◽

Inner Membrane ◽

Protoporphyrinogen Oxidase ◽

N Terminus ◽

Membrane Spanning

Protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), the penultimate enzyme of haem biosynthesis, is a nucleus-encoded flavoprotein strongly associated with the outer surface of the inner mitochondrial membrane. It is attached to this membrane by an unknown mechanism that appears not to involve a membrane-spanning domain. The pathway for its import to mitochondria and insertion into the inner membrane has not been established. We have fused human PPOXs containing N-terminal deletions, C-terminal deletions or missense mutations to yellow fluorescent protein (YFP) and have used these constructs to investigate the mitochondrial import of PPOX in human cells. We show that all the information required for efficient import is contained within the first 250 amino acid residues of human PPOX and that targeting to mitochondria is prevented by fusion of YFP to the N-terminus. Deletion of between 151 and 175 residues from the N-terminus is required to abolish import, whereas shorter deletions impair its efficiency. Fully efficient targeting appears to require both a major targeting signal, the whole or part of which is contained between residues 151 and 175, and which may be involved in anchoring to the inner mitochondrial membrane, together with interaction between this region and a sequence(s) within the first 150 residues. These features suggest that the mechanism for import of human PPOX to mitochondria differs from those identified for the translocation of nucleus-encoded, membrane-spanning, inner membrane proteins. In addition, a missense mutation outside this region (Val335→Gly) prevented targeting to mitochondria and delayed the appearance of YFP fluorescence. This mutation appeared to prevent import by a direct effect on protein folding rather than by altering a sequence required for targeting. It may lead to sequestration of the PPOX–YFP construct in an unfolded conformation, followed by proteolytic degradation, possibly through enhanced binding to a cytosolic chaperone protein.

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Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0806338105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15376-15381 ◽

Cited By ~ 134

Author(s):

Mark C. Leake ◽

Nicholas P. Greene ◽

Rachel M. Godun ◽

Thierry Granjon ◽

Grant Buchanan ◽

...

Keyword(s):

Single Molecule ◽

Protein Transport ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Protonmotive Force ◽

Oligomeric State ◽

Essential Components ◽

Tat System ◽

Folded Proteins

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of ≈25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains ≈15 mobile TatA complexes and a pool of ≈100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

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Direct Interaction of the Novel Nox Proteins with p22phox Is Required for the Formation of a Functionally Active NADPH Oxidase

Journal of Biological Chemistry ◽

10.1074/jbc.m406486200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45935-45941 ◽

Cited By ~ 376

Author(s):

Rashmi K. Ambasta ◽

Pravir Kumar ◽

Kathy K. Griendling ◽

Harald H. H. W. Schmidt ◽

Rudi Busse ◽

...

Keyword(s):

Nadph Oxidase ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Direct Interaction ◽

Cell Types ◽

Hek293 Cells ◽

Anion Formation ◽

Co Precipitation

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.

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Applicability of superfolder YFP bimolecular fluorescence complementation in vitro

Biological Chemistry ◽

10.1515/bc.2009.008 ◽

2009 ◽

Vol 390 (1) ◽

Cited By ~ 18

Author(s):

Corinna Ottmann ◽

Michael Weyand ◽

Alexander Wolf ◽

Jürgen Kuhlmann ◽

Christian Ottmann

Keyword(s):

Protein Interactions ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Bimolecular Fluorescence Complementation ◽

Structural Basis ◽

Protein Protein Interactions ◽

Fluorescence Complementation ◽

Bimolecular Fluorescence

Abstract Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) is a widely employed method to study protein-protein interactions in cells. As yet, this technique has not been used in vitro. To evaluate a possible application of BiFC in vitro, we constructed a ‘superfolder split YFP’ system where 15 mutations enhance expression of the fusion proteins in Escherichia coli and enable a native purification due to improved solubility. Here, we present the crystal structure of ‘superfolder YFP’, providing the structural basis for the enhanced folding and stability characteristics. Complementation between the two non-fluorescent YFP fragments fused to HRas and Raf1RBD or to 14-3-3 and PMA2-CT52 resulted in the constitution of the functional fluorophore. The in vivo BiFC with these protein interaction pairs was demonstrated in eukaryotic cell lines as well. Here, we present for the first time BiFC in vitro studies with natively purified superfolder YFP fusion proteins and show the potential and drawbacks of this method for analyzing protein-protein interactions.

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FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts

Biochemical Journal ◽

10.1042/bj20091388 ◽

2010 ◽

Vol 425 (3) ◽

pp. 567-573 ◽

Cited By ~ 63

Author(s):

Stephanie Engel ◽

Silvia Scolari ◽

Bastian Thaa ◽

Nils Krebs ◽

Thomas Korte ◽

...

Keyword(s):

Influenza Virus ◽

Plasma Membrane ◽

Fluorescent Protein ◽

Chinese Hamster Ovary Cells ◽

Yellow Fluorescent Protein ◽

Strong Association ◽

Chinese Hamster ◽

Membrane Rafts ◽

Fluorescence Lifetime Imaging ◽

Targeting Signals

It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA–Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA–Cer with Myr-Pal–YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal–YFP exhibited a much faster mobility compared with HA–Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.

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Identification and Regulation of Genes for Cobalamin Transport in the Cyanobacterium Synechococcus sp. Strain PCC 7002

Journal of Bacteriology ◽

10.1128/jb.00476-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2753-2761 ◽

Cited By ~ 19

Author(s):

Adam A. Pérez ◽

Dmitry A. Rodionov ◽

Donald A. Bryant

Keyword(s):

Active Transport ◽

Transport System ◽

In Silico ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Transport Systems ◽

Uptake System ◽

Reporter System ◽

Content Type ◽

Active Transport System

ABSTRACTThe cyanobacteriumSynechococcussp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis ofl-methionine by methionine synthase (MetH).Synechococcussp. strain PCC 7002 is unable to synthesize cobalaminde novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticatedin silicoprediction tools, abtuB-cpdA-btuC-btuFoperon encoding components putatively required for a B12uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by high-throughput RNA sequencing of a cobalamin-independent form ofSynechococcussp. strain PCC 7002 grown in the absence or presence of cobalamin confirmed regulation of thebtuoperon by cobalamin. Pérez et al. (A. A. Pérez, Z. Liu, D. A. Rodionov, Z. Li, and D. A. Bryant, J Bacteriol 198:2743–2752, 2016,http://dx.doi.org/10.1128/JB.00475-16) developed a cobalamin-dependent yellow fluorescent protein reporter system in aSynechococcussp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of thebtuuptake system by assessing the ability of targeted mutants to transport cobalamin. ThebtuBpromoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to ayfpreporter. The combined data indicate that thebtuB-cpdA-btuF-btuCoperon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch.IMPORTANCEWith a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacteriumSynechococcussp. strain PCC 7002. This study demonstrates the importance of experimental validation ofin silicopredictions and provides a general scheme forin vivoverification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified inSynechococcussp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of thebtuoperon that encodes the components of the cobalamin active-transport system.

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