No abstract

Quantitative ultrastructural analysis and proteomics detail CLIC structure, composition, and function.

SummaryClathrin-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.

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.

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.

Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the ␣-and ␤2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.The family name sorting nexin (SNX) 1 is given to a large group of proteins that are represented throughout the eukaryotic kingdom. Sorting nexins are characterized by the presence of a subclass of the phosphoinositide-binding phox (PX) domain, and it is believed that a common function of proteins in this family is to participate in sorting processes in the cell. Several members localize to endosomal structures, and some of the SNX proteins have been shown to directly interact with transmembrane receptors to regulate their sorting in the endosomal pathway (for a review, see Ref. 1). The localization of SNX proteins is suggested to be determined by their PX domains, although protein-protein interactions may also contribute to the membrane specificity (2, 3).SNX9 and its close relative SNX18 are the only members of the sorting nexin family that contains an Src homology 3 (SH3) domain. SH3 domains interact specifically with short prolinecontaining sequences (PXXP) and are present in a large number of proteins (4). Target sequences are often located in distinct domains, referred to as proline-rich domains (PRDs). SNX9 was originally identified as a molecule that interacted with PRDs in certain metalloprotease disintegrins (ADAMs) (5). Work in Drosophila showed that SNX9 (named DSH3PX1) interacted with Dock (the fly orthologue of mammalian Nck) and Dscam (Down's syndrome cell adhesion molecule) to form a complex involved in axonal guidance in the fly (6, 7). In addition, SNX9 was found to interact with the clathrin-binding tyrosine kinase Ack through an SH...

The molecular mechanisms implicated in endosomal sorting are currently attracting much attention and they have turned out to be more complex than first realized (Bonifacino and Rojas, 2006;Maxfield and McGraw, 2004). A number of different factors are required to support the sorting decisions that have to be made to ensure that different cargoes are taken to the appropriate destinations according to the needs of the cell. Transport intermediates can either be in the form of round vesicles or extended tubules, and some cells display an elaborate system of endocytic membrane networks that are thought to be involved in specific trafficking events. The dynamic organelles commonly referred to as early endosomes appear to be major sorting stations within the system, and can be functionally divided into sorting endosomes and recycling endosomes. Internalized material can either recycle back to the plasma membrane by one of several routes, be sorted to the transGolgi network (TGN) or end up in the lysosomal pathway leading to degradation of the cargo.Membrane-binding and -modulating proteins are important for the mechanics of the endocytic system. Classical examples are the vesicular coat proteins, such as clathrin and its adaptor proteins (APs) (Edeling et al., 2006b;Robinson, 2004;Ungewickell and Hinrichsen, 2007). Clathrin acts as a vesicular stabilizing protein at several locations in the cell, and clathrin-dependent carrier formation requires membrane-specific APs for localization, coat assembly, accessory protein recruitment and cargo selection. There are four different kinds of APs, which operate at diverse cellular sites. For example, whereas AP-2 is mostly found at the plasma membrane participating in clathrin-dependent endocytosis, AP-1 is concentrated at the TGN to mediate clathrin-dependent trafficking of cargo to the endosomal system. AP-1 is also found on endosomal membranes and is proposed to take part in trafficking pathways for retrieval of cargo to the TGN. However, it is unclear how the same AP can work in trafficking pathways that go in opposite directions. An arrangement like this would probably require different procedures for carrier formation, to give unique identities of the transport intermediates. Such differences might be mediated by specific accessory proteins and membrane-modulating proteins. The advantage of using the same cargo-binding adaptor for anterograde and retrograde traffic is that efficient recycling systems can be created for proteins that shuttle between compartments, such as the mannose 6-phosphate receptor and furin.Proteins that can bind and modulate specific membrane zones are important players in the process of vesicular-and tubular-carrier formation. Examples are proteins with phosphoinositide-binding PX (phox homology) and PH (pleckstrin homology) domains (Di Paolo and De Camilli, 2006;Lemmon, 2003), as well as proteins with membrane-bending function (Zimmerberg and Kozlov, 2006). A class of proteins with the latter property is the recently described BAR (Bin/Amphiphysin/...

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.