The retromer is a trimeric cargo-recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson's disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer's subcellular localization and its capacity to sort cargo. We show that expression of the PD-linked Vps35 D620N mutant redistributes retromer-positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild-type Vps35. While PD-linked point mutant Vps35 D620N interacts with the cation-independent mannose-6-phosphate receptor (CI-M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI-M6PR ligand and protease responsible for degradation of α-synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro-and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kg) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kg function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kg do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.endosome | protein crystallography | X-ray scattering | membrane trafficking T he cell-surface levels of signaling and adhesion receptors, nutrient transporters, ion channels, and many other proteins are tightly regulated by opposing endocytic, exocytic, and endosomal recycling transport pathways. The selective sorting of these transmembrane proteins is the consequence of their interaction with essential adaptor proteins via conserved motifs present in their cytosolic tails, generally based on short linear amino acid sequences such as the Yxxϕ, DxxLL, and [DE]xxxL [LI] motifs (where ϕ is any bulky hydrophobic side-chain, and x is any residue) recognized by clathrin adaptors (1-3). The first identified sorting signal was the Asn-Pro-Xaa-Tyr (NPxY) motif, initially described in the LDL receptor (LDLR) isolated from patients suffering from familial hypercholesterolemia, where mutation of the sequence results in defective internalization and cholesterol uptake (4). The recent structure of the autosomal recessive hypercholesterolemia protein (ARH) phosphotyrosinebinding (PTB) domain in complex with the LDLR intracellular domain (ICD) provides the molecular basis of LDLR recognition and internalization within clathrin-coated pits by endocytic adaptor proteins (5).Although little is known about the sorting sequences and mechanisms required for competing endosome-to-cell surface recycling pathways, emerging evidence shows that the NPxY motif not only is vital to internalization but also aids cargo recycling via organelle-specific recognition by the endosomal protein, sorting nexin 17 (SNX17). SNX17 is a member of the Phox-homology (PX) domain-containing protein family and has been shown to be a critical regulator of endosomal sorting and cell-surface recycling of several essential cargo molecules. These include P-selectin (6-8), amyloid precursor protein (APP) (9, 10), integrins (11,12), and members of the LDL receptor family (13-17) ...

†These authors contributed equally to this work.Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.

Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3 P , others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3 P , non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3 P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.