First published online 2 March 2005
doi: 10.1242/dev.01716
Development 132, 1699-1711 (2005)
Published by The Company of Biologists 2005
VAN3 ARF–GAP-mediated vesicle transport is involved in leaf vascular network formation
Koji Koizumi1,*,
,
Satoshi Naramoto2,,
Shinichiro Sawa2,,
Natsuko Yahara3,
Takashi Ueda2,
Akihiko Nakano2,3,
Munetaka Sugiyama4 and
Hiroo Fukuda2,5,
1 Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of
Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
2 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Molecular Membrane Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan
4 Botanical Gardens, Graduate School of Science, The University of Tokyo, 3-7-1
Hakusan, Bunkyo-ku, Tokyo 112-0001, Japan
5 Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi,
Kanagawa 230-0045, Japan
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Fig. 5. Characterization of the VAN3 protein. (A,B) ARF–GAP activity of
recombinant VAN3 proteins in vitro. GAP activity was assessed by the
hydrolysis of GTP bound to the yeast ARF1 protein. (A) Concentration
dependence of VAN3. Recombinant VAN3 protein was serially diluted, and
incubated with 1 µM [ -32P]GTP-loaded ARF protein at
30°C for 10 minutes. (B) Time-course of ARF–GAP activity of VAN3.
Recombinant VAN3 (1 µM) was incubated with 1 µM
[-32P]GTP-loaded ARF1 protein at 30°C. Aliquots were
withdrawn at the indicated times. (C) Fat western blots of phospholipids
probed with recombinant VAN3. Phospholipids are indicated above each blot, and
the amount of lipid spotted onto the nitrocellulose is shown to the left of
each row of lipid. The blot was incubated with 0.5 µg/ml GST-tagged
recombinant type V VAN3. PA, phosphatidic acid; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PI-4-P,
phosphatidylinositol 4-monophosphate; PI-4,5-P2, phosphatidylinositol
4,5-bisphosphate. (D) Homodimerization of VAN3 through the BAR domain.
Constructs including full-length VAN3 (FLVAN3) and seven types of
truncated VAN3 were fused to GAL4 AD in pGADT7, and those
containing FLVAN3 and type I VAN3 were fused to GAL4 DNA-BD
in pGBKT7. Proteins are represented by bars; motifs or domains within proteins
are indicated by different colors. Circles show protein–protein
interactions. Crosses show no interaction.
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Fig. 1. Vein patterns in van3-related mutants. Vein patterns of the
cotyledons of 7-day-old seedlings (A,B,E,F,G,H,K,L) and in first-node leaves
of 11-day-old plants (C,D,I,J). (A) Wild-type. (B) van3 mutant. (C)
Wild-type. (D) van3 mutant. (E) pin1-3 mutant. (F) van3
pin1-3 double mutant. (G) emb30-7/gn mutant. (H)
van3 emb30-7/gn double mutant. (I)
emb30-7/gn mutant. (J) van3 emb30-7/gn
double mutant. (K) mpT370 mutant. (L) van3 mpT370 double
mutant. Genotypes were identified by CAPS analysis using root DNA. Scale bars:
(A-L) 1 mm.
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Fig. 2. Effects of NPA and BFA on vein patterning. Vein patterns of first-node
leaves of the wild-type (A-D,I-L) and the van3 mutant (E-H,M-P) grown
for 11 days on agar medium containing NPA (A-H) or BFA (I-P). Mock-treated
leaves are shown in A,E,I,M. Chemicals were applied at concentrations of 5
µM (B,F,J,N), 10 µM (C,G,K,O), or 20 µM (D,H,L,P). Inset in L is a
close-up of a trachery element island under Nomarski optics. Scale bars:
(A-P), 500 µm.
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Fig. 3. DR5::GUS expression patterns. DR5 expression patterns in
the developing first-node leaves of the wild-type (A-D) and van3
mutant (F-I). Samples were harvested from 6-(A,F), 7-(B,G), 8-(C,H) and
10-day-old (D,I) seedlings. E and J show magnified views of the boxed regions
in C and G, respectively. DR5::GUS expression pattern in the excised
cotyledons (K-P) and first-node leaves (Q-V) treated with NAA. The wild-type
(K-M,Q-S) and van3 mutant (N-P,T-V) samples were exposed to NAA for 1
hour: mock-treated controls (K,N,Q,T); 1 µM NAA (L,O,R,U), 10 µM NAA
(M,P,S,V). Scale bars: 100 µm.
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Fig. 4. Molecular cloning and expression of VAN3. (A) VAN3 was
isolated by positional cloning and mapped to the 89 kb region between the
T31B5c and T22N19c markers, which corresponds to two adjoining BAC clones,
T31B5 and T22N19. Numbers below the molecular markers indicate the
recombination frequency between the marker and the VAN3 locus
(recombinant chromosomes/analyzed chromosomes). Black lines and outlines show
the VAN3 gene (At5g13300) and putative genes in this region,
respectively. (B) Structure of the VAN3 protein and its homologs from the
predicted proteins of Arabidopsis and humans. The VAN3 gene
encodes an ARF–GTPase-activating protein (ARF–GAP) that includes
BAR, PH, ARF–GAP and three-ANK-repeat domains. The mutation in the
van3 mutant (TGG [Trp356] to TAG [stop]) is indicated. (C) Comparison
of BAR and ARF–GAP domains. Conserved amino acid residues are
highlighted by grey boxes. (D) Semi-quantitative RT–PCR analysis of the
expression of the VAN3 gene in the wild-type and van3
mutant. RNA was isolated from 7-day-old seedling cotyledons of the wild-type
and van3 mutant that had been either mock-treated (0) or treated with
10 µM NAA for 1 hour (10). ACT2 is the control. Numbers of cycles
of PCR are indicated on the right.
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Fig. 6. Subcellular localization of VAN3. (A) Arabidopsis
suspension-cultured cells. (B-D,G-I) Location of VAN3–Venus (red) and
GFP-tagged subcellular marker genes (green). 35S-promoter-driven Venus-tagged
VAN3 and GFP-tagged subcellular markers were co-introduced into protoplasts of
Arabidopsis suspension-cultured cells. (B-D) Merged image of
VAN3–Venus and the ER marker HDEL–GFP (B), the cis-Golgi marker
SYP31–GFP (C), the endosome marker ARA6–GFP (D). (E) Location of
ARA7–GFP in the gnom mutant cells. (F) VAN3–Venus in
gnom mutant cells. (G–I) Localization of the TGN marker
SYP41–GFP (G) and VAN3–Venus (H), and a merged image of G and H
(I). E and F are projection images of serial confocal planes, A-D and G-I and
single confocal slice images. Scale bars: (A-I) 5 µm.
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Fig. 7. Models of the functional differentiation of TGN in vesicle trafficking
pathways. The TGN does not have a uniform function but is functionally
differentiated into subpopulations that are specialized to transport to the
plasma membranes (yellow), to endosomes (blue), or to prevacuolar compartments
(PVC) (white). VAN3 is located in a subpopulation of the TGN that may be
involved in the transport of auxin signaling modules and/or secreted vascular
formation factors.
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© The Company of Biologists Ltd 2005
