Regulation of endosomal membrane traffic by a Gadkin/AP-1/kinesin KIF5 complex

Abstract

Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed ␥-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)-and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.motor-protein driven transport ͉ clathrin adaptor AP-1 ͉ endosomal vesicles ͉ recycling T he endosomal system comprises a mosaic of dynamically interconnected organelles that fulfills a variety of important cell physiological functions ranging from the uptake, recycling, and degradation of nutrients, signaling molecules and cell surface receptors to the regulation of cell migration, differentiation, and morphogenesis (1-3). The endocytic pathway also intersects with the biosynthetic delivery of lysosomal enzymes at several stations, most notably at the trans-Golgi network (TGN)/ endosomal boundary (1). Endosomes and TGN-or endosomederived vesicles exhibit characteristic distribution patterns with Rab4-positive sorting endosomal vesicles (EVs) and tubular recycling endosomes (REs) typically concentrated in the pericentrosomal area (4). Early endosomes, by contrast, appear dispersed throughout the cytoplasm (5). Function and dynamics of endosomes and EVs requires a so far ill-defined interplay between organellar sorting adaptors, the cytoskeleton (6) and molecular motors (7). Here we show that ␥-BAR, a recently described accessory factor of the clathrin adaptor complex AP-1 at the TGN/endosomal interface (8), modulates the dynamics of transferrin (Tf)-and Rab4-positive EVs by directly associating with AP-1 and kinesin KIF5. Because ␥-BAR does not harbor a curvature-sensing BIN/amphiphysin/Rvs167 (BAR) domain, we refer to this protein as Gadkin, for ␥1-adaptin and kinesin interactor. The AP-1/Gadkin/KIF5 complex identified here provides a hitherto unknown molecular link between TGN-derived EVs and the microtubule-based cytoskeleton. Our work also suggests a surprising complexity of endosomal membrane dynamics and its integration with cargo sorting. Results Gadkin Localizes to the TGN and to Perinuclear EVs and Regulates EVPositioning. Gadkin has originally been identified as an AP-1 binding protein localized to the T...

A Novel Subtype of AP-1-binding Motif within the Palmitoylated trans-Golgi Network/Endosomal Accessory Protein Gadkin/γ-BAR

“…Overexpressed WT Gadkin localized to peripheral puncta (Fig. 1C) corresponding to Tfand AP-1-positive TGN-derived endosomal vesicles (EVs) that accumulate in the periphery due to the tight association of Gadkin with the plus-end-directed microtubule motor protein kinesin KIF5 (19). A similar dispersion of AP-1-and Tf-positive endosomes was also observed upon overexpression of c-myc-or FLAG-tagged Gadkin (data not shown).…”

“…The precise mapping of the AP-1 binding determinants described here should pave the way for a more thorough investigation of these possibilities. We have recently shown that Gadkin directly binds to the light chains of kinesin KIF5 via a site non-overlapping with any of the AP-1 binding determinants identified in the present work (19). This positions Gadkin at the interface of AP-1-mediated sorting of transmembrane cargo and transport of TGN-derived EVs along the microtubule-based cytoskeleton.…”

“…The precise mechanism underlying this activity of ␥-BAR has not been revealed. We have recently shown that ␥-BAR directly interacts with the light chains of kinesin KIF5, thereby contributing to the peripheral movement of TGN-derived endosomal vesicles (19). Because ␥-BAR does not harbor a curvature-sensing BAR (BIN/amphiphysin/ Rvs167) domain, we propose to refer to this protein as Gadkin, for ␥1-adaptin and kinesin interactor, to avoid possible confusion with BAR domain proteins.…”

Kinesin-1 promotes post-Golgi trafficking of NCAM140 and NCAM180 to the cell surface

“…Confirming the physiological role of this association, NCAM could be co-immunoprecipitated with KLC1 from mouse brain tissue lysates. The KLC1-binding motif within the intracellular domain of NCAM140 and NCAM180 comprises the amino acid sequence CGKAGPGA and does not contain tyrosine or tryptophan residues such as have been shown to be crucial for the interaction between KLC1 and other cargoes (Aoyama et Hayakawa et al, 2007;Konecna et al, 2006;Rosa-Ferreira and Munro, 2011;Schmidt et al, 2009;Verhey et al, 2001). Therefore, the identified sequence in NCAM represents a new binding motif for KLC1.…”

On the move: endocytic trafficking in cell migration

“…Accordingly, the loss of AP-2 results in decreased microtubule acetylation as well as reduced directionality of 2D and invasive migration [59]. Another adaptor, the AP- !-associated protein Gadk:in [60], that regulates recycling endosomal traffic by linking AP-1 positive vesicles to the microtubule-dependent motor protein kinesin 1 [61], was likewise found to have a complementary role in cell migration. Gadkin binds additionally to the actin nucleator ARP2/3, which is crucial for Jamellipodia formation.…”