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

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.

Download Full-text

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

Biochemistry and Cell Biology ◽

10.1139/o09-066 ◽

2009 ◽

Vol 87 (6) ◽

pp. 963-974 ◽

Cited By ~ 7

Author(s):

David J. McKinnon ◽

Pawel Brzezowski ◽

Kenneth E. Wilson ◽

Gordon R. Gray

Keyword(s):

Isocitrate Dehydrogenase ◽

Fusion Proteins ◽

Protein Transport ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Transport Systems ◽

Amino Acid Residues ◽

Active Protein ◽

Nicotiana Tabacum L ◽

Targeting Signals

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.

Download Full-text

Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111281119 ◽

2022 ◽

Vol 119 (1) ◽

pp. e2111281119

Author(s):

Hsi-En Tsao ◽

Shu Nga Lui ◽

Anthony Hiu-Fung Lo ◽

Shuai Chen ◽

Hiu Yan Wong ◽

...

Keyword(s):

Fluorescent Protein ◽

Storage Proteins ◽

Salt Bridges ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

C Terminus ◽

Vacuolar Sorting ◽

Structural Insights ◽

Cargo Binding

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Modified Bean Seed Protein Phaseolin Did Not Accumulate Stably in Transgenic Tobacco Seeds after Methionine Enhancement Mutations

American Journal of Plant Sciences ◽

10.4236/ajps.2015.65069 ◽

2015 ◽

Vol 06 (05) ◽

pp. 640-650 ◽

Cited By ~ 1

Author(s):

Eric Lasserre ◽

T. S. Ko ◽

John M. Dyer ◽

Norimoto Murai

Keyword(s):

Transgenic Tobacco ◽

Seed Protein ◽

Bean Seed ◽

Tobacco Seeds

Download Full-text

Biogenesis of the Protein Storage Vacuole Crystalloid

The Journal of Cell Biology ◽

10.1083/jcb.150.4.755 ◽

2000 ◽

Vol 150 (4) ◽

pp. 755-770 ◽

Cited By ~ 116

Author(s):

Liwen Jiang ◽

Thomas E. Phillips ◽

Sally W. Rogers ◽

John C. Rogers

Keyword(s):

Seed Protein ◽

Animal Cells ◽

Reporter Protein ◽

Developmentally Regulated ◽

Protein Storage ◽

Protein Storage Vacuoles ◽

Protein Storage Vacuole ◽

Vacuolar System ◽

Unique Mechanism ◽

Storage Vacuole

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.

Download Full-text