Role of SH3 domain-containing proteins in clathrin-mediated vesicle trafficking in Arabidopsis.

A group of plant AtSH3Ps (Arabidopsis thaliana SH3-containing proteins) involved in trafficking of clathrin-coated vesicles was identified from the GenBank database. These proteins contained predicted coiled-coil and Src homology 3 (SH3) domains that are similar to animal and yeast proteins involved in the formation, fission, and uncoating of clathrin-coated vesicles. Subcellular fractionation and immunolocalization studies confirmed the presence of AtSH3P1 in the endomembrane system. In particular, AtSH3P1 was localized on or adjacent to the plasma membrane and its associated vesicles, vesicles of the trans-Golgi network, and the partially coated reticulum. At all of these locations, AtSH3P1 colocalized with clathrin. Functionally, in vitro lipid binding assay demonstrated that AtSH3P1 bound to specific lipid groups known to accumulate at invaginated coated pits or coated vesicles. In addition, immunohistochemical studies and actin binding assays indicated that AtSH3P1 also may regulate vesicle trafficking along the actin cytoskeleton. Yeast complementation studies suggested that AtSH3Ps have similar functions to the yeast Rvs167p protein involved in endocytosis and actin arrangement. A novel interaction between AtSH3P1 and an auxilin-like protein was identified by yeast two-hybrid screening, immunolocalization, and an in vitro binding assay. The interaction was mediated through the SH3 domain of AtSH3P1 and a proline-rich domain of auxilin. The auxilin-like protein stimulated the uncoating of clathrin-coated vesicles by Hsc70, a reaction that appeared to be inhibited in the presence of AtSH3P1. Hence, AtSH3P1 may perform regulatory and/or scaffolding roles during the transition of fission and the uncoating of clathrin-coated vesicles.