Tobacco RhoGTPase ACTIVATING PROTEIN1 Spatially Restricts Signaling of RAC/Rop to the Apex of Pollen Tubes

Abstract

Regulation by Rho-type small GTPases, such as RAC5, is important for the maintenance of polarity in tobacco (Nicotiana tabacum) pollen tubes. We previously showed that RhoGDI2 is necessary for RAC5 localization. Here, we describe the GTPase activating protein RhoGAP1 that controls the area of RAC5 activity. RhoGAP1 N-terminal and CRIB (for Cdc42/Racinteractive binding) domains are both necessary for targeting yellow fluorescent protein-RhoGAP1 fusions to the plasma membrane close to, but not in, pollen tube apices. We propose that this localization restricts apical Rho-type GTPase activity from spreading toward the flanks, which ensures the maintenance of RAC signaling at the apex. The CRIB domain is not required but enhances in vitro RhoGAP1 activity toward the pollen tube-specific-RAC5. A mutation reducing GAP activity of RhoGAP1 leads to ballooning pollen tubes resembling those overexpressing RAC5. To ascertain the specific targeting mechanism of RhoGAP1, we isolated a 14-3-3 protein interacting with RhoGAP1. When overexpressed with RhoGAP1, it counteracts the growth-retarding effect of RhoGAP1 overexpression and attenuates RhoGAP1 membrane localization but, overexpressed alone, induces only small architectural changes. We propose that inactivation of RAC5 by the subapically localized RhoGAP1, together with dynamic relocalization of inactivated RAC5 from flanks to tip by RhoGDI2, leads to spatial restriction of RAC5 to pollen tube apices, thereby sustaining polar growth.

Type B Phosphatidylinositol-4-Phosphate 5-Kinases MediateArabidopsisandNicotiana tabacumPollen Tube Growth by Regulating Apical Pectin Secretion

“…The presence of a ring-like arrangement of cortical actin, termed the actin fringe, near the growing pollen tube tip has been demonstrated to be essential for reverse-fountain streaming, and pharmacological perturbation of actin fringe assembly results in the loss of directional vesicle traffic and abolishes cell polarity (Cardenas et al, 2008). Other elements participating in the regulation of tip growth in plant cells include a variety of essential regulatory GTPases (Kost et al, 1999;Preuss et al, 2004;Gu et al, 2005;Klahre and Kost, 2006;Song et al, 2006) as well as factors required for vesicle trafficking and targeted secretion of cargo vesicles (Yuen et al, 2005;Song et al, 2006).…”

A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells

“…In Arabidopsis thaliana, there are at least eight ROPGAPlike proteins that can be subdivided in two classes depending on whether they have a CDC42/RAC-interactive binding (CRIB) domain for G-protein binding or a pleckstrin homology domain for lipid binding (Wu et al, 2000;Eklund et al, 2010). ROPGAPs locally regulate ROP signaling in cell polarity by switching off active ROPs (Klahre and Kost, 2006;Hwang et al, 2008Hwang et al, , 2010.…”

Regulation of pollen tube polarity

“…RopGAPs accelerate the intrinsic GTPase activity of Rop, thereby preventing the lateral diffusion of activated Rop during tube growth. 9,10 The localization and activity of RopGAPs are in turn regulated by Rop through a CRIB domain (Cdc42/Rac interactive binding motif ) within RopGAPs, which facilitates Rop-binding and promotes GAP activity. 9,10 RopGDIs sequester GDP-bound Rop in the cytosol where GTP is abundant.…”

“…9,10 The localization and activity of RopGAPs are in turn regulated by Rop through a CRIB domain (Cdc42/Rac interactive binding motif ) within RopGAPs, which facilitates Rop-binding and promotes GAP activity. 9,10 RopGDIs sequester GDP-bound Rop in the cytosol where GTP is abundant. 2 Because RopGDIs preferentially bind to prenylated, GDP-bound Rop at the plasma membrane, 11 between the apical and subapical plasma membrane during pollen tube growth, and by doing so, restrict the membrane distribution of Rop to the apical region.…”