SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

SummaryEarly endosome-to-trans-Golgi network (TGN) transport is organized by the retromer complex. Consisting of cargo-selective and membrane-bound subcomplexes, retromer coordinates sorting with membrane deformation and carrier formation. Here, we describe four mammalian retromers whose membrane-bound subcomplexes contain specific combinations of the sorting nexins (SNX), SNX1, SNX2, SNX5, and SNX6. We establish that retromer requires a dynamic spatial organization of the endosomal network, which is regulated through association of SNX5/SNX6 with the p150glued component of dynactin, an activator of the minus-end directed microtubule motor dynein; an association further defined through genetic studies in C. elegans. Finally, we also establish that the spatial organization of the retromer pathway is mediated through the association of SNX1 with the proposed TGN-localized tether Rab6-interacting protein-1. These interactions describe fundamental steps in retromer-mediated transport and establish that the spatial organization of the retromer network is a critical element required for efficient retromer-mediated sorting.

The mammalian retromer is a multimeric protein complex involved in mediating endosome-to-trans-Golgi-network retrograde transport of the cation-independent mannose-6-phosphate receptor. The retromer is composed of two subcomplexes, one containing SNX1 and forming a membrane-bound coat, the other comprising VPS26, VPS29 and VPS35 and being cargo-selective. In yeast, an additional sorting nexin - Vps17p - is a component of the membrane bound coat. It remains unclear whether the mammalian retromer requires a functional equivalent of Vps17p. Here, we have used an RNAi loss-of-function screen to examine whether any of the other 30 mammalian sorting nexins are required for retromer-mediated endosome-to-trans-Golgi-network retrieval of the cation-independent mannose-6-phosphate receptor. Using this screen, we identified two proteins, SNX5 and SNX6, that, when suppressed, induced a phenotype similar to that observed upon suppression of known retromer components. Whereas SNX5 and SNX6 colocalised with SNX1 on early endosomes, in immunoprecipitation experiments only SNX6 appeared to exist in a complex with SNX1. Interestingly, suppression of SNX5 and/or SNX6 resulted in a significant loss of SNX1, an effect that seemed to result from post-translational regulation of the SNX1 level. Such data suggest that SNX1 and SNX6 exist in a stable, endosomally associated complex that is required for retromer-mediated retrieval of the cation-independent mannose-6-phosphate receptor. SNX5 and SNX6 may therefore constitute functional equivalents of Vps17p in mammals.

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX⌽, where ⌽ represents a bulky hydrophobic residue. This motif interacts with the medium chain ( ) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXX⌽ motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXX⌽ motifs and the 2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073-28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosinebinding pocket in 2 provides a structural explanation for A23 being able to inhibit the interaction between YXX⌽ motifs and 2. Furthermore, we show that A23 inhibited the internalization of 125 I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXX⌽ motif/ 2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXX⌽ motif/ 2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.The trafficking and intracellular targeting of many transmembrane proteins depend on motifs found within their cytosolic domains. One such motif contains a critical tyrosine residue within the sequence YXX⌽, where ⌽ represents a bulky hydrophobic residue (1, 2). Tyrosine-based motifs conforming to this consensus sequence can interact directly with the medium chain ( ) subunits of heterotetrameric adaptor complexes involved in several different intracellular trafficking pathways. The AP-2 adaptor complex facilitates incorporation of transmembrane proteins containing YXX⌽ motifs into clathrincoated vesicles at the cell surface (1, 3-9), whereas AP-1, AP-3, and AP-4 adaptor complexes are involved in other intracellular vesicular transport steps (reviewed in Refs. 10 -13). The subunits from all four adaptor complexes have been shown to interact with the YXX⌽ motif, with the precise sequence and context of this motif determining the specificity of the interaction (14, 15). Mutation of residues within and around the YXX⌽ motif affect interaction with subunits; but, in all cases, mutation of the tyrosine residue in the motif (e.g. to alanine) blocks interaction with the subunit (4, 6, 8). Structural studies on peptides containing these motifs have generated data that hav...

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXX+(+ represents a bulky hydrophobic residue). This motif interacts with the medium chain (mu) subunits of adaptor complexes which link the cytosolic domain of the integral membrane protein to the clathrin coat involved in vesicle formation. The motif YXX+ is similar to motifs which are phosphorylated by tyrosine kinases. Tyrphostins (structural analogues of tyrosine) are inhibitors of tyrosine kinases: can they inhibit interactions between mu chains and YXX+ motifs? We have investigated several tyrphostins and found that, in vitro and in the yeast two hybrid system, some are capable of inhibiting the intraction between YXX+ motifs and different medium chains. We have also shown that tyrphostins which block these interactions affect the internalisation and recyling of the transferrin receptor in live cells.At present little is known regarding the mechanism of metabotopic glutamate receptor (mGluR) trafficking. In order to study this, we inserted a hemagglutinin (HA) epitope tag between residues 57-58 of the extracellular domain of the rat mGluRla splice variant. In HEK293 cells transiently transfected with HA-mGlula, the epitope tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (1OpM; 30 min) the HA-mGluRla internalized to early endosomes and an endocytic recycling compartment. Further quantification of receptor endocytosis was provided by ELISA experiments which showed that the HA-mGluRla underwent rapid agonist-(glutamate 10 inpM) induced internalization with a t o,5 of 19 ? 3 min. To determine whether agonist-induced mGluRla internalization is an arrestin-and dynamin-dependent process cells were cotransfected with HA-mGluRla and either dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant construct with HA-mGluRla strongly inhibited glutmate-induced (10 pM; 30 min) internalization. In addition arrestin-2-GFP or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluRla. Taken together our observations demonstrate that agonist-induced internalization of mGluRla is an arrestin-and dynamin-dependent process.Arrestin-and Dynamin-dependent. 73The effect of pertussis toxin on the internalisation of the Somatostatin agonists are rapidly and efficiently endocytosed with the somatostatin2 receptor. The aim of this study was to determine the role of G protein coupling on internalisation by examining the effect of pertussis toxin treatment. The accumulation of internalised agonists and receptors is governed by a number of processes including hormone-receptor association, dissociation, receptor endocytosis and recycling back to the plasma membrane. We have established a mathematical model describing these processes and used this model in the mechanistic interpretat...