In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2m or AP2s partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2s, but not AP2m, was required for SA-and tyrphostin A23-dependent inhibition of CME. Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells.

Although exocytosis is critical for the proper trafficking of materials to the plasma membrane, relatively little is known about the mechanistic details of post-Golgi trafficking in plants. Here, we demonstrate that the DENN (Differentially Expressed in Normal and Neoplastic cells) domain protein STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) and SCD2 form a previously unknown protein complex, the SCD complex, that functionally interacts with subunits of the exocyst complex and the RabE1 family of GTPases in Consistent with a role in post-Golgi trafficking, and mutants display defects in exocytosis and recycling of PIN2-GFP. Perturbation of exocytosis using the small molecule Endosidin2 results in growth inhibition and PIN2-GFP trafficking defects in and mutants. In addition to the exocyst, the SCD complex binds in a nucleotide state-specific manner with Sec4p/Rab8-related RabE1 GTPases and overexpression of wild-type RabE1 rescues temperature-sensitive mutants. Furthermore, SCD1 colocalizes with the exocyst subunit, SEC15B, and RabE1 at the cell plate and in distinct punctae at or near the plasma membrane. Our findings reveal a mechanism for plant exocytosis, through the identification and characterization of a protein interaction network that includes the SCD complex, RabE1, and the exocyst.

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PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild

The root is an ideal model system for studying subcellular localization and dynamic trafficking of important membrane-associated proteins in plants. Immunofluorescence analysis is necessary to reveal subcellular localization and intracellular trafficking of endogenous proteins as epitope tags or fluorescent proteins may cause mislocation of fusion proteins. Here, we describe a rapid and reliable immunodetection protocol for whole-mount in situ localization of membrane-associated proteins involved in clathrin-mediated endocytosis (CME) in Arabidopsis root cells. The whole procedure includes five basic steps: tissue fixation, tissue permeation, blocking, primary antibody incubation, and secondary antibody incubation.