The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Yamauchi, Seiji; Fusada, Naoki; Hayashi, Hidenori; Utsumi, Toshihiko; Uozumi, Nobuyuki; Endo, Yaeta; Tozawa, Yuzuru

doi:10.1111/j.1742-4658.2010.07768.x

Cited by 34 publications

(29 citation statements)

References 40 publications

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“…We confirmed that the wheat germ lysates supported N-terminal myristoylation of Gag-YFP (Fig. 1A), as reported previously (34), and that in liposome flotation assays, Gag-YFP synthesized in wheat germ lysates bound to liposomes in a PI(4,5)P 2 -dependent manner (Fig. 1B).…”

Section: Efficient Binding Of Hiv-1 Gag-yfp To Guvs Requires Treatmensupporting

confidence: 74%

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Olety

Veatch

Ono

2015

J Virol

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HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P 2 -specific pleckstrin homology domain of phospholipase C␦1 (PH PLC␦1 ), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P 2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P 2 with unsaturated or saturated acyl chains affect membrane binding of PH PLC␦ 1 and Gag. Both unsaturated dioleoyl-PI(4,5)P 2 [DO-PI(4,5)P 2 ] and saturated dipalmitoyl-PI(4,5)P 2 [DP-PI(4,5)P 2 ] successfully recruited PH PLC␦ 1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P 2 but not DP-PI(4,5)P 2 recruited Gag to GUVs, indicating that PI(4,5)P 2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P 2 , cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PH PLC␦ 1 regardless of PI(4,5)P 2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P 2 had DO-acyl chains. DP-PI(4,5)P 2 -containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P 2 still promoted recruitment of Gag, but not PH PLC␦ 1 , to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P 2 acyl chains in membrane binding of two PI(4,5)P 2 -binding proteins, Gag and PH PLC␦ 1 . Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P 2 containing an unsaturated acyl chain over DP-PI(4,5)P 2 , suggesting that Gag sensitivity to PI(4,5)P 2 acyl chain saturation is determined directly by the matrix-PI(4,5)P 2 interaction, rather than indirectly by a myristate-dependent mechanism. IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P 2 . Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P 2 -interacting domains represented by PH PLC␦ 1 . However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P 2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P 2 with an unsaturated acyl chain recruited PH PLC␦ 1 and Gag similarly, whereas PI(4,5)P 2 with saturated acyl chains either recruited PH PLC␦ 1 but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P 2 acyl chains differentially modulate membrane binding of PI(4,5)P 2 -binding proteins. Since Gag membrane binding is essential for progeny...

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Section: Efficient Binding Of Hiv-1 Gag-yfp To Guvs Requires Treatmensupporting

confidence: 74%

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Olety

Veatch

Ono

2015

J Virol

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“…For these reactions, a plasmid encoding Gag-YFP under the control of a T7 promoter is used. Of note, as shown earlier, wheat germ lysates support myristoylation without additional components 23,24 . Using this method, it has been possible to obtain sufficient quantities of full-length myristoylated Gag-YFP for visualization of Gag on GUV membranes 24 .…”

Section: Introductionsupporting

confidence: 79%

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

Olety

Veatch

Ono

2016

JoVE

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The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a "cell-like" environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated fulllength Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy. Video LinkThe video component of this article can be found at

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“…Myristoylator (Bologna et al, 2004) and The MYR Predictor (http://mendel.imp.ac.at/myristate/ SUPLpredictor.htm) predicted N-myristoylation sites for LOG2 and LUL1-4. Moreover, in vitro myristoylation of LUL1 was recently demonstrated (Yamauchi et al, 2010). To experimentally verify that LOG2 could be myristoylated, LOG2 and the corresponding G2A mutant (bearing a substitution known to inhibit N-terminal myristoylation; Gordon et al, 1991) were expressed in rabbit reticulocyte lysate-coupled transcription-translation systems in the presence of [ 3 H]myristic acid or [ 3 H]Leu.…”

Section: Myristoylation Of Log2 Is Important For Its Membrane Localizmentioning

confidence: 99%

The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for GLUTAMINE DUMPER1-Induced Amino Acid Secretion in Arabidopsis

et al. 2012

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Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

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The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Cited by 34 publications

References 40 publications

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for GLUTAMINE DUMPER1-Induced Amino Acid Secretion in Arabidopsis

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