The Clathrin Adaptor Complex AP-2 Mediates Endocytosis of BRASSINOSTEROID INSENSITIVE1 inArabidopsis 

Abstract

Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptormediated endocytosis.

Helping Hands for Budding Prospects: ENTH/ANTH/VHS Accessory Proteins in Endocytosis, Vacuolar Transport, and Secretion

“…Various mutants in AP-2 subunits show impaired CME and developmental aberrations that presumably are the consequence of these cellular defects. These results, plus the dynamic spatiotemporal localization of AP-2 components at the PM, support the notion that AP-2 is an early adaptor complex for clathrin recruitment/assembly (Di Rubbo et al, 2013;Fan et al, 2013;Kim et al, 2013;Yamaoka et al, 2013) in plant endocytosis.…”

The dynamics of plant plasma membrane proteins: PINs and beyond

“…4A). Pharmacological and dominant-negative approaches using the C-terminal HUB domain of CHC, which competes for binding with CLCs, interfere with CME in plant cells (Dhonukshe et al, 2007), and the analysis of Arabidopsis clathrin loss-of-function mutants confirmed these results and revealed a crucial role for CME in bulk endocytosis, with a particular focus on PIN proteins (Dhonukshe et al, 2007;Kitakura et al, 2011;Di Rubbo et al, 2013). Moreover, and in accordance with a requirement for endocytosis in the polarization of de novo synthesized PIN1 and PIN2, clathrin appears necessary for the proper establishment of PIN polarity (KleineVehn et al, 2011).…”

“…Arabidopsis clathrin chains associate with AP2 subunits and form punctate foci at the plasma membrane (Fan et al, 2013;Kim et al, 2013;Yamaoka et al, 2013). As such, and similar to clathrin mutants, plants impaired in AP2 subunits show alterations in general endocytosis and PIN internalization and/or polarity, as well as defects in the endocytosis of BRI1, which correlates with severe developmental defects (Di Rubbo et al, 2013;Fan et al, 2013;Kim et al, 2013;Yamaoka et al, 2013). More recently, the activity of another, apparently plant-specific complex -the TPLATE adaptor complex -has been linked to early events in CME.…”

“…Moreover, and in accordance with a requirement for endocytosis in the polarization of de novo synthesized PIN1 and PIN2, clathrin appears necessary for the proper establishment of PIN polarity (KleineVehn et al, 2011). Besides PINs, several other plasma membrane proteins, such as the iron transporter IRT1, the boron transporter BOR1, BRI1, and the aquaporin water channel PIP2, are also dependent on clathrin for their internalization (Dhonukshe et al, 2007;Takano et al, 2010;Barberon et al, 2011;Irani et al, 2012;Di Rubbo et al, 2013). In line with such a general role in endocytic sorting, genetic interference with clathrin function in plants results in severe developmental defects, including alterations in root elongation, root and hypocotyl gravitropism, and lateral root primordia initiation Wang et al, 2013).…”

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

“…It is now known that endocytosis and recycling play important roles in cell wall remodeling, gravitropism, pathogen defense, guard cell movement, and hormone signaling in plants (Samaj et al, 2005). Recently, many clathrin-mediated endocytosis (CME) components and cargo proteins have been identified and characterized in Arabidopsis thaliana (Konopka et al, 2008;Van Damme et al, 2011;Ito et al, 2012;Song et al, 2012;Bashline et al, 2013;Di Rubbo et al, 2013;Fan et al, 2013;Kim et al, 2013;Wang et al, 2013;Yamaoka et al, 2013;Gadeyne et al, 2014). One particular cargo protein, cellulose synthase (CESA), acts as a unique anchor that can be used to dissect endocytic uptake and recycling pathways in plants (Bashline et al, , 2014a.…”