Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P2 bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P2 bound to the RLD1.