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.
Highlights
Endosomes are a primary “vehicle” for protein transport within the cell (Jovic et al, 2010)
The multi-domain-containing protein, GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1), is involved in a variety of processes including membrane-shaping, small Gprotein signaling, clathrin-independent endocytosis and cell spreading (Doherty and Lundmark, 2009). Because it contains both a BAR and PH domain, and has a role in membrane bending similar to that of other BAR-containing proteins, and because it localizes to tubular endosomes (Lundmark et al, 2008), we posited that GRAF1 may be involved in the biogenesis of tubular recycling endosomes (TREs)
Since MICAL-L1- and Rab11containing recycling endosomes (RE) are target-vesicles for GRAF1, we hypothesized that GRAF1-induced vesiculation might directly affect the exit of cargo molecules from the recycling compartment either to the plasma membrane (PM), or to the intracellular degradative pathways. β1 integrins are transmembrane proteins that mediate the attachment of the cells to the extracellular matrix
Summary
Endosomes are a primary “vehicle” for protein transport within the cell (Jovic et al, 2010) This transportation includes the sorting and delivering of cargo molecules to their corresponding destinations, which depends upon a series of well-coordinated membrane fission and fusion events. A large group of cytosolic proteins assemble onto membranes to influence their shape, and facilitate the formation of a budding vesicle These vesicles/carriers are transported along microtubule tracks to a specific destination where they eventually fuse with a target membrane and release their cargo. Such BAR domaincontaining proteins induce membranes into highly curved shapes (McMahon and Gallop, 2005; Zimmerberg and Kozlov, 2006) and include the N-BAR protein Amphiphysin/Bin (Pant et al, 2009), and the F-BAR protein Syndapin, known as PACSIN2 (Braun et al, 2005; Giridharan et al, 2013)
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