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First published online April 10, 2009
doi: 10.1242/10.1242/dev.030098

Development 136, 1529-1538 (2009)
Published by The Company of Biologists 2009
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Phosphoinositide-dependent regulation of VAN3 ARF-GAP localization and activity essential for vascular tissue continuity in plants

Satoshi Naramoto1,2,3,{dagger}, Shinichiro Sawa1, Koji Koizumi1,*, Tomohiro Uemura1, Takashi Ueda1, Jírí Friml3, Akihiko Nakano1,2 and Hiroo Fukuda1,{dagger}

1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
2 Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.
3 Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, 9052 Gent, Belgium.


Figure 1
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  		Fig. 1. Illustration of the positions of VAN3 mutations and the venation patterns of van3-related mutants. (A) Diagram of the VAN3 gene structure and the alignment of sequences of the PH domain of VAN3 and its homologues. Arrowheads indicate nonsense, frameshift or deletion mutations that lead to a strong van3 phenotype. Arrows indicate missense mutations. (B-J) Vein patterns of cotyledons. Wild type (B), a van3-1 mutant (C), a van3-2 mutant (D), a van3-1/van3-2 mutant (E), a cvp2 mutant (F), a cvp2cvl1 mutant (G), a van3-2cvp2 mutant (H), a vab mutant (I) and a van3-2vab mutant (J). (K-N) Vein patterns of first leaves. Wild type (K), a van3-2 mutant (L), a vab mutant (M) and a van3-2vab mutant (N). Scale bars: 1 mm.

 

Figure 2
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  		Fig. 2. Subcellular localization of VAN3. (A) Overview of the functional VAN3-VENUS fusion construct used. The positions indicated are relative to the translational start site. The VENUS tag was translationally fused to the 3' end of the ORF of the genomic XbaI-SpeI fragment. (B-G) Localization of GFP-tagged organelle markers (green) and VENUS-tagged VAN3 (red) in young leaf petioles. Localization of the TGN marker GFP-SYP41 (B), VAN3-VENUS (C) and their merged image (D). Merged images of VAN3-VENUS with GFP-SYP41 (E), with the Golgi body marker ST-GFP (F) and with the PVC marker GFP-ARA7/RAB-F2b (G). The inset of E indicates an enlarged view of the TGN-localized VAN3-VENUS. The arrowheads show the TGN-localized VAN3-VENUS and the arrows show the VAN3-VENUS signal that is not marked by GFP-SYP41. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. The involvement of the BAR and PH domain in the subcellular localization of VAN3. (A) Schematic representation of deletion mutant proteins of VAN3. (B-H) Confocal images of the root vascular cells expressing sGFP-tagged VAN3 deletion mutant proteins under the control of the authentic promoter. BAR-PH-ARFGAP-sGFP (B), BAR-PH-sGFP (C), BAR-sGFP (D), PH-ARFGAP-3xANK-sGFP (E), ARFGAP-3xANK-sGFP (F), 3xANK-sGFP (G), and PH-sGFP (H). Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Interaction of VAN3 with CVP2, CVL1 and VAB. (A-C) Subcellular localization of VAN3-sGFP (A), CVP2-eYFP (B) and their merged image (C). (D-F) Subcellular localization of VAN3-sGFP in wild-type (D), cvp2 (E) and cvp2cvl1 plants (F). (G-I) Subcellular localization of VAN3G321E-sGFP in wild-type (G), cvp2 (H) and vab (I) plants. (J,K) Subcellular localization of VAN3-sGFP in vab (J) and cvp2vab (K) plants. Scale bars: 10 µm in A-C; 50 µm in D-K.

 

Figure 5
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  		Fig. 5. Molecular characterization of VAB. (A) Schematic representation of VAB structure. (B) Deduced amino acid sequence of VAB. Underlining indicates the region of the PH domain. (C-E) Subcellular localization of VAN3-sGFP (C) and VAB-eYFP (D), and their merged image (E). (F-I) BiFC analysis of VAN3 and VAB. (F,G) YFP fluorescence (F) and DIC images (G) of protoplasts co-transformed with VAN3-nYFP and VAB-cYFP. (H,I) YFP fluorescence (H) and DIC images (I) of protoplasts co-transformed with VAL3-nYFP and VAB-cYFP. Scale bars: 10 µm in C-E; 5 µm in F-I.

 

Figure 6
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  		Fig. 6. Effect of phosphoinositides and IP3 on the ARF-GAP activity of VAN3. VAN3-dependent GTP hydrolysis in myristoylated, GTP-loaded AtArf1 was measured in the presence of phosphoinositides and IP3. GTP hydrolysis was initiated by the addition of 50 nM of GST-VAN3 and 100 nM of the indicated phosphoinositides, and the nucleotide exchange in the AtArf1 was monitored as the change in autofluorescence of intrinsic tryptophan. (A) Phosphoinositide-dependent ARF GAP activity. Different phospholipids or inositol phosphate were added with AtArf1 and VAN3. (B) The domain of VAN3 required for ARF GAP activity. Mutated VAN3 proteins were added instead of VAN3.

 

Figure 7
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  		Fig. 7. A model of the 5PTase-mediated VAN3 regulation. At5PTase, including CVP2/CVL1 may increase PtdIns(4)P level or decrease IP3 level or both. PtdIns(4)P may have a dual function of recruiting VAN3 to the TGN membranes and promoting the ARF-GAP activity of VAN3, whereas IP3 may inhibit these processes. This regulation by PtdIns(4)P and IP3 may occur through their association with the PH domain of VAN3. VAB may assist VAN3 in binding to the PtdIns(4)P-rich subdomain of the TGN membrane.

 

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© The Company of Biologists Ltd 2009