title: GRAF1 forms a complex with MICAL-L1 and EHD1 to cooperate in tubular recycling endosome vesiculation.
abstract: The biogenesis of tubular recycling endosomes (TREs) and their subsequent vesiculation after cargo-sorting has occurred, is essential for receptor and lipid recycling to the plasma membrane. Although recent studies have implicated the C-terminal Eps15 Homology Domain (EHD) protein, EHD1, as a key regulator of TRE vesiculation, additional proteins involved in this process have been largely uncharacterized. In the present study, we identify the GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1) protein in a complex with EHD1 and the TRE hub protein, Molecules Interacting with CasL-Like1 (MICAL-L1). Over-expression of GRAF1 caused vesiculation of MICAL-L1-containing TRE, whereas GRAF1-depletion led to impaired TRE vesiculation and delayed receptor recycling. Moreover, co-addition of purified EHD1 and GRAF1 in a semi-permeabilized cell vesiculation assay produced synergistic TRE vesiculation. Overall, based on our data, we suggest that in addition to its roles in clathrin-independent endocytosis, GRAF1 synergizes with EHD1 to support TRE vesiculation.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, The University of Nebraska Medical Center Omaha, NE, USA.
authors.forename: Bishuang
authors.initials: B
authors.lastname: Cai
authors.affiliation: Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, The University of Nebraska Medical Center Omaha, NE, USA.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Xie
authors.affiliation: Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, The University of Nebraska Medical Center Omaha, NE, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Caplan
authors.affiliation: Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, The University of Nebraska Medical Center Omaha, NE, USA.
authors.forename: Naava
authors.initials: N
authors.lastname: Naslavsky
keywords: BAR domain; EHD1; GRAF1; MICAL-L1; PH domain; tubular recycling endosome; tubulation; vesiculation