Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting.

Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This "PIP-stop" releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.

Secondary structure and (1)H, (13)C, (15)N resonance assignments of the endosomal sorting protein sorting nexin 3.

Sorting nexin 3 (SNX3) belongs to a sub-family of sorting nexins that primarily contain a single Phox homology domain capable of binding phosphoinositides and membranes. We report the complete (1)H, (13)C and (15)N resonance assignments of the full-length human SNX3 protein and identification of its secondary structure elements, revealing a canonical fold and unstructured termini.

Structural basis of dynamic membrane recognition by trans-Golgi network specific FAPP proteins.

Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking.

Molecular characterization of Myxobolus cuttacki (Myxozoa, Myxosporea, Bivalvulida) infecting gill lamellae of minor carp Labeo bata (Ham.).

As new pathogenic strains are emerging and threatening aquaculture development, myxosporeans (Myxozoa) are receiving much attention in recent years. Myxosporean taxonomy is traditionally based on morphology of the myxospore stage. Molecular data on Indian myxosporeans are rare. In this report, the 18S rRNA gene sequence of Myxobolus cuttacki infecting gill lamellae of minor carp Labeo bata (Ham.) and its phylogenetic relationship with other myxobolids are described for the first time. The plasmodia of M. cuttacki were 0.5-0.9 mm in size and whitish with a round to oval shape. The mean mature spore size was 16.10×7.05 µm. The 18S rRNA nucleotide sequence with 1703 bp of M. cuttacki (accession number KF465682) clustered phylogenetically with other Myxobolus spp. infecting cyprinid gills with 78-90% homogeneity. The gill lamellae infecting M. catmrigalae (KC933944) and M. orissae (KF448527) of Indian major carp Cirrhinus mrigala from India, exhibited 86% and 81% homogeneity with M. cuttacki, respectively. The infection rate was low to moderate on the gills which can have a negative impact on respiratory and physiological functions and subsequently on fish production.

Binding to syntenin-1 protein defines a new mode of ubiquitin-based interactions regulated by phosphorylation.

Syntenin-1 is a PDZ domain-containing adaptor that controls trafficking of transmembrane proteins including those associated with tetraspanin-enriched microdomains. We describe the interaction of syntenin-1 with ubiquitin through a novel binding site spanning the C terminus of ubiquitin, centered on Arg(72), Leu(73), and Arg(74). A conserved LYPSL sequence in the N terminus, as well as the C-terminal region of syntenin-1, are essential for binding to ubiquitin. We present evidence for the regulation of this interaction through syntenin-1 dimerization. We have also established that syntenin-1 is phosphorylated downstream of Ulk1, a serine/threonine kinase that plays a critical role in autophagy and regulates endocytic trafficking. Importantly, Ulk1-dependent phosphorylation of Ser(6) in the LYPSL prevents the interaction of syntenin-1 with ubiquitin. These results define an unprecedented ubiquitin-dependent pathway involving syntenin-1 that is regulated by Ulk1.

Structure and function of BamE within the outer membrane and the ß-barrel assembly machine.

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential ß-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.

Secondary structure and 1H, 13C and 15N resonance assignments of BamE, a component of the outer membrane protein assembly machinery in Escherichia coli.

We report the (1)H, (13)C and (15)N backbone and side chain chemical shift assignments and secondary structure of the Escherichia coli protein BamE, a subunit of the BAM (Omp85) complex, the ß-barrel assembly machinery present in all Gram-negative bacteria, mitochondria and chloroplasts and is essential for viability.

Secondary structure and (1)H, (13)C and (15)N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli.

We report the (1)H, (13)C and (15)N backbone chemical shift assignments and secondary structure of the Escherichia coli protein BamC, a 32-kDa protein subunit that forms part of the BAM (Omp85) complex, the beta-barrel assembly machinery present in all Gram-negative bacteria and which is essential for viability.