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

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.

Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking inArabidopsis

“…Furthermore, CME has been linked to phosphoinositides in tobacco (Nicotiana tabacum) pollen tubes (Zhao et al, 2010). To further substantiate the hypothesis that CME of PIN proteins requires PtdIns(4,5)P 2 in Arabidopsis, we monitored the dynamic membrane association of GFP-tagged CLATHRIN LIGHT CHAIN 2 (CLC2-GFP) (Wang et al, 2013) in 7-d-old roots of wild-type controls and pip5k1 pip5k2 double mutants by spinning disc confocal live imaging (Figure 9; see Supplemental Movies 1 and 2 online). In wild-type controls, CLC2-GFP decorated small punctae in the focal plane of the plasma membrane and larger, fast-moving structures outside the focal plane likely representing the trans-Golgi network (Figure 9A; see Supplemental Movies 1 and 2 online).…”

The dynamics of plant plasma membrane proteins: PINs and beyond

“…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). 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).…”

Lotus japonicus Clathrin Heavy Chain1 Is Associated with Rho-Like GTPase ROP6 and Involved in Nodule Formation

“…Clathrin-mediated endocytosis is a very well-conserved process in animal and plant cells and involves highly homologous protein components, including CHC, CLC, adaptor protein complex2, and the eight-core component protein complex, TPLATE (Pérez-Gómez and Moore, 2007; Kitakura et al, 2011;Di Rubbo et al, 2013;Kim et al, 2013;Wang et al, 2013a, Gadeyne et al, 2014. CHCs are highly conserved (.90% similarity) in the plant kingdom.…”

“…A control root sample was performed in the absence of the primary antibody. The anti-CHC polyclonal antibody, generated against a conserved peptide (FNELISLMESGLGLERAHMG; Wang et al, 2013a) that is present in AtCHC, LjCHC1, and LjCHC2, was provided by Jianwei Pan.…”