Clathrin Light Chains Regulate Clathrin-Mediated Trafficking, Auxin Signaling, and Development inArabidopsis

Chao Wang,Wei Fu,Jianwei Pan,Xu Yan,Sebastian Y Bednarek,Nan Jiang,Chuanyou Li,Qian Chen,Jianzhong Liu,Bojun Ma

doi:10.1105/tpc.112.108373

Abstract

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.

Highlights

Clathrin-mediated endocytosis (CME) is an evolutionally conserved endocytic pathway that plays a pivotal role in the regulation of protein abundance at the plasma membrane (PM) and/ or the trans-Golgi network (TGN) during signaling events and retargeting or degradation of membrane proteins (Chen et al, 2011; McMahon and Boucrot, 2011)
Previous studies have demonstrated that CLC2-GFP localizes to the TGN/ EE and to dynamic PM-associated foci (Konopka et al, 2008; Ito et al, 2012), and upon auxin treatment, the levels of CLC2-GFP transiently disappear from the PM but remain constant at the trans-Golgi network/early endosome (TGN/EE) (Robert et al, 2010)
In contrast with the results of Robert et al (2010), the association of CLC2-GFP with the TGN/EE paralleled the auxin-stimulated loss, followed by recovery of the PM pool of CLC2-GFP

Summary

Introduction

Clathrin-mediated endocytosis (CME) is an evolutionally conserved endocytic pathway that plays a pivotal role in the regulation of protein abundance at the plasma membrane (PM) and/ or the trans-Golgi network (TGN) during signaling events and retargeting or degradation of membrane proteins (Chen et al, 2011; McMahon and Boucrot, 2011). Clathrin is a triskelionshaped complex consisting of three heavy chains (CHCs), which form the structural backbone of the clathrin lattice, and three light chains (CLCs) (Royle, 2006), which have been suggested in mammalian and yeast cells to regulate the formation of clathrincoated vesicles (CCVs) (Ybe et al, 1998; Newpher et al, 2006; Royle, 2006; Mettlen et al, 2009). Plant CLCs and CHCs predominantly localize to the PM and TGN/early endosome (EE) compartments (Dhonukshe et al, 2007; Konopka et al, 2008; Robert et al, 2010; Ito et al, 2012), and CME regulates the internalization of critical PM proteins involved in hormone signaling (Geldner et al, 2007; Robert et al, 2010; Xu et al, 2010; Irani et al, 2012), defense or stress responses (LeborgneCastel et al, 2008), and nutrient uptake (Barberon et al, 2011).

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