Molecular chaperon HSP70-9 is required for clathrin-mediated endocytosis in Arabidopsis

Clathrin-mediated endocytosis (CME) plays a key role in the internalization of plasma membrane-localized proteins, lipids and extracellular substances; however, the regulatory mechanism of CME in plants is unclear. In this work, we demonstrated that molecular chaperon HSP70-9 is required for CME in Arabidopsis thaliana. Knocking out the HSP70-9 gene led to auxin-related phenotypes and the accumulation of auxin efflux carrier PINs in root cells, indicating that HSP70-9 is involved in the PIN trafficking process. Immunolocalization studies showed that the HSP70-9 was localized in mitochondria, vesicles and cytoplasm. Next, we found that HSP70-9 and clathrin light chain 1 (CLC1) were co-localized in cells, and there was a strong interaction between the two. HSP70-9 knockout led to an increase in CLC1-related vesicle number in root cells, and the application of protein synthesis inhibitor cycloheximide did not significantly inhibit the increase in the hsp70-9a mutant. HSP70-9 knockout did not significantly affect the level of CLC1 mRNA, but reduced the abundance of free CLC1 protein in root cells, indicating that HSP70-9 might be involved in the stability of CLC1-related vesicles. Moreover, our data indicated that the introduction of the exogenous CLC1 gene rescued the hsp70-9a seedlings likely through promoting PIN trafficking. Furthermore, the role of HSP70-9 in the depolymerization of clathrin-coated vesicles (CCVs) was investigated in vitro. The result indicated that the addition of HSP70-9 promoted the release of CLC1 from the CCVs. Taken together, our data suggest that HSP70-9 affects PIN trafficking likely through facilitating the release of CLC1 from the CCVs.