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An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region.

Hashimoto K, Igarashi H, Mano S, Takenaka C, Shiina T, Yamaguchi M, Demura T, Nishimura M, Shimmen T, Yokota E - J. Exp. Bot. (2008)

Bottom Line: Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner.Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue.It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1279, Japan.

ABSTRACT
Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

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Quantitative analysis of His6-AtRab proteins bound to MYA2 tail 1 fused with GST in in vitro binding assays. (A) Binding of His6-AtRab proteins to MYA2 tail 1 coated beads in the absence (–GTP) or presence of GTP (+GTP). White and shaded bars show the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellet with MYA2 tail 1 coated beads. Each AtRab protein was mixed with beads in the absence or presence of GTP, or with control beads in the presence of GTP. After centrifugation, the pellets were subjected to SDS-PAGE, and the intensity of each AtRab protein band was determined. The intensities are shown as a relative value to that of control beads. Averages ±SD from three separate experiments are plotted. (B) Dissociation of His-AtRab proteins from MYA2 tail 1 by washing with a buffer containing GTP. White and shaded bars showed the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellets with MYA2 tail 1 coated beads. Each His-AtRab protein was mixed with MYA2 tail 1 coated beads in the presence or absence of GTP. The beads were washed twice as described in the Materials and methods. The amounts of each AtRab protein recovered in the first pellets (P1), second pellets (P2), and third pellets (P3) are given relative to that of pellets in the absence of GTP. Averages ±SD from three separate experiments are plotted.
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fig3: Quantitative analysis of His6-AtRab proteins bound to MYA2 tail 1 fused with GST in in vitro binding assays. (A) Binding of His6-AtRab proteins to MYA2 tail 1 coated beads in the absence (–GTP) or presence of GTP (+GTP). White and shaded bars show the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellet with MYA2 tail 1 coated beads. Each AtRab protein was mixed with beads in the absence or presence of GTP, or with control beads in the presence of GTP. After centrifugation, the pellets were subjected to SDS-PAGE, and the intensity of each AtRab protein band was determined. The intensities are shown as a relative value to that of control beads. Averages ±SD from three separate experiments are plotted. (B) Dissociation of His-AtRab proteins from MYA2 tail 1 by washing with a buffer containing GTP. White and shaded bars showed the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellets with MYA2 tail 1 coated beads. Each His-AtRab protein was mixed with MYA2 tail 1 coated beads in the presence or absence of GTP. The beads were washed twice as described in the Materials and methods. The amounts of each AtRab protein recovered in the first pellets (P1), second pellets (P2), and third pellets (P3) are given relative to that of pellets in the absence of GTP. Averages ±SD from three separate experiments are plotted.

Mentions: First, the interaction of both recombinant AtRab proteins with the MYA2 tail 1 was examined. In the absence of GTP (lane b in Fig. 2B), the amount of both His6-AtRab proteins, which recovered in pellets with beads coated with the MYA2 tail 1, appeared to be same as that recovered with control beads (lane a in Fig. 2B). However, the amount of both AtRab proteins in pellets with MYA2 tail 1 coated beads was increased by the addition of GTP (lane c in Fig. 2B). Densitometric band analysis of AtRab protein in the pellet showed that the amount of AtRabD1 (white bars in Fig. 3A) and AtRabC2a protein (shaded bars in Fig. 3A) bound to the MYA2 tail 1 coated beads in the presence of GTP (+GTP in Fig. 3A) was 2.6-fold and 1.7-fold, respectively, larger than that in the absence of GTP (–GTP in Fig. 3A) or that bound to the control beads in the presence of GTP, indicating that both recombinant AtRab proteins had an ability to bind to the MYA2 tail 1 in a GTP-dependent manner and that the affinity of AtRabD1 for the MYA2 tail 1 was higher than that of AtRabC2a.


An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region.

Hashimoto K, Igarashi H, Mano S, Takenaka C, Shiina T, Yamaguchi M, Demura T, Nishimura M, Shimmen T, Yokota E - J. Exp. Bot. (2008)

Quantitative analysis of His6-AtRab proteins bound to MYA2 tail 1 fused with GST in in vitro binding assays. (A) Binding of His6-AtRab proteins to MYA2 tail 1 coated beads in the absence (–GTP) or presence of GTP (+GTP). White and shaded bars show the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellet with MYA2 tail 1 coated beads. Each AtRab protein was mixed with beads in the absence or presence of GTP, or with control beads in the presence of GTP. After centrifugation, the pellets were subjected to SDS-PAGE, and the intensity of each AtRab protein band was determined. The intensities are shown as a relative value to that of control beads. Averages ±SD from three separate experiments are plotted. (B) Dissociation of His-AtRab proteins from MYA2 tail 1 by washing with a buffer containing GTP. White and shaded bars showed the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellets with MYA2 tail 1 coated beads. Each His-AtRab protein was mixed with MYA2 tail 1 coated beads in the presence or absence of GTP. The beads were washed twice as described in the Materials and methods. The amounts of each AtRab protein recovered in the first pellets (P1), second pellets (P2), and third pellets (P3) are given relative to that of pellets in the absence of GTP. Averages ±SD from three separate experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2561144&req=5

fig3: Quantitative analysis of His6-AtRab proteins bound to MYA2 tail 1 fused with GST in in vitro binding assays. (A) Binding of His6-AtRab proteins to MYA2 tail 1 coated beads in the absence (–GTP) or presence of GTP (+GTP). White and shaded bars show the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellet with MYA2 tail 1 coated beads. Each AtRab protein was mixed with beads in the absence or presence of GTP, or with control beads in the presence of GTP. After centrifugation, the pellets were subjected to SDS-PAGE, and the intensity of each AtRab protein band was determined. The intensities are shown as a relative value to that of control beads. Averages ±SD from three separate experiments are plotted. (B) Dissociation of His-AtRab proteins from MYA2 tail 1 by washing with a buffer containing GTP. White and shaded bars showed the amount of His-AtRabD1 and His-AtRabC2a, respectively, in the pellets with MYA2 tail 1 coated beads. Each His-AtRab protein was mixed with MYA2 tail 1 coated beads in the presence or absence of GTP. The beads were washed twice as described in the Materials and methods. The amounts of each AtRab protein recovered in the first pellets (P1), second pellets (P2), and third pellets (P3) are given relative to that of pellets in the absence of GTP. Averages ±SD from three separate experiments are plotted.
Mentions: First, the interaction of both recombinant AtRab proteins with the MYA2 tail 1 was examined. In the absence of GTP (lane b in Fig. 2B), the amount of both His6-AtRab proteins, which recovered in pellets with beads coated with the MYA2 tail 1, appeared to be same as that recovered with control beads (lane a in Fig. 2B). However, the amount of both AtRab proteins in pellets with MYA2 tail 1 coated beads was increased by the addition of GTP (lane c in Fig. 2B). Densitometric band analysis of AtRab protein in the pellet showed that the amount of AtRabD1 (white bars in Fig. 3A) and AtRabC2a protein (shaded bars in Fig. 3A) bound to the MYA2 tail 1 coated beads in the presence of GTP (+GTP in Fig. 3A) was 2.6-fold and 1.7-fold, respectively, larger than that in the absence of GTP (–GTP in Fig. 3A) or that bound to the control beads in the presence of GTP, indicating that both recombinant AtRab proteins had an ability to bind to the MYA2 tail 1 in a GTP-dependent manner and that the affinity of AtRabD1 for the MYA2 tail 1 was higher than that of AtRabC2a.

Bottom Line: Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner.Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue.It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1279, Japan.

ABSTRACT
Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

Show MeSH
Related in: MedlinePlus