Richard Lundmark

Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling

The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

The GTPase-Activating Protein GRAF1 Regulates the CLIC/GEEC Endocytic Pathway

Summary Clathrin-independent endocytosis is an umbrella term for a variety of endocytic pathways that internalize numerous cargoes independently of the canonical coat protein Clathrin [1, 2]. Electron-microscopy studies have defined the pleiomorphic CLathrin-Independent Carriers (CLICs) and GPI-Enriched Endocytic Compartments (GEECs) as related major players in such uptake [3, 4]. This CLIC/GEEC pathway relies upon cellular signaling and activation through small G proteins, but mechanistic insight into the biogenesis of its tubular and tubulovesicular carriers is lacking. Here we show that the Rho-GAP-domain-containing protein GRAF1 marks, and is indispensable for, a major Clathrin-independent endocytic pathway. This pathway is characterized by its ability to internalize bacterial exotoxins, GPI-linked proteins, and extracellular fluid. We show that GRAF1 localizes to PtdIns(4,5)P2-enriched, tubular, and punctate lipid structures via N-terminal BAR and PH domains. These membrane carriers are relatively devoid of caveolin1 and flotillin1 but are associated with activity of the small G protein Cdc42. This study provides the first specific noncargo marker for CLIC/GEEC endocytic membranes and demonstrates how GRAF1 can coordinate small G protein signaling and membrane remodeling to facilitate internalization of CLIC/GEEC pathway cargoes.

The PX-BAR membrane-remodeling unit of sorting nexin 9

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin‐mediated endocytosis in non‐neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane‐remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide‐containing membranes. The study provides insights into the SNX9‐induced membrane modulation mechanism.

Regulated membrane recruitment of dynamin-2 mediated by sorting nexin 9.

The endocytic proteins sorting nexin 9 (SNX9) and dynamin-2 (Dyn2) assemble in the cytosol as a resting complex, together with a 41-kDa protein. We show here that the complex can be activated for membrane binding of SNX9 and Dyn2 by incubation of cytosol in the presence of ATP. SNX9 was essential for Dyn2 recruitment, whereas the reverse was not the case. RNA interference experiments confirmed that SNX9 functions as a mediator of Dyn2 recruitment to membranes in cells. The 41-kDa component was identified as the glycolytic enzyme aldolase. Aldolase bound with high affinity to a tryptophan-containing acidic sequence in SNX9 located close to its Phox homology domain, thereby blocking the membrane binding activity of SNX9. Phosphorylation of SNX9 released aldolase from the native cytosolic complex and rendered SNX9 competent for membrane binding. The results suggest that SNX9-dependent recruitment of Dyn2 to the membrane is regulated by an interaction between SNX9 and aldolase.

Arf family GTP loading is activated by, and generates, positive membrane curvature

Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.

EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization.

EH domain-containing 2 (EHD2) specifically and stably associates with caveolae at the plasma membrane and interacts with pacsin2 and cavin1. A loop in the nucleotide-binding domain, together with ATP binding, is required for caveolar localization. EHD2 stabilizes caveolae at the surface to control their dynamics.

SNX9 - a prelude to vesicle release.

The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking.

SNX9, SNX18 and SNX30 constitute a separate subfamily of PX-BAR-containing sorting nexin (SNX) proteins. We show here that most tissues express all three paralogs, and immunoprecipitation and immunofluorescence experiments demonstrated that the SNX9-family proteins act as individual entities in cells. Their SH3 domains displayed a high selectivity for dynamin 2, and the PX-BAR units had the capacity to tubulate membranes when expressed in HeLa cells. As previously described for the PX-BAR domain of SNX9 (SNX9-PX-BAR), purified SNX18-PX-BAR caused liposome tubulation in vitro and had a binding preference for PtdIns(4,5)P2. However, contrary to SNX9, which primarily acts in clathrin-mediated endocytosis at the plasma membrane, endogenous SNX18 localized to AP-1- and PACS1-positive endosomal structures, which were devoid of clathrin and resistant to Brefeldin A. Moreover, a γ-adaptin recognition motif was defined in a low-complexity region of SNX18, and a complex of endogenous SNX18 and AP-1 could be immunoprecipitated after Brefeldin A treatment. Overexpression of SNX18 sequestered AP-1 from peripheral endosomes and resulted in the formation of short SNX18-decorated tubes with distinct dynamin puncta. The results indicate that SNX9-family members make up discrete membrane-scission units together with dynamin, and suggest that SNX18 mediates budding of carriers for AP-1-positive endosomal trafficking.

The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2

Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

EspF of enteropathogenic Escherichia coli binds sorting nexin 9.

EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.