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

SUMMARY 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.