Characterization of GABA B receptor-like proteins in Dictyostelium discoideum

GPCRs are a superfamily of diverse integral membrane proteins that allow detection and transduction of a large variety of extracellular signals. In Dictyostelium only seven members of one family, the crl (cAMP receptor like) family had been identified and studied in detail. The analysis of the genome sequence uncovered 48 additional putative GPCRs grouped into the secretin (family 2), metabotropic glutamate/GABAB (family 3) and the frizzled / smoothened (family 5) families of receptors. The presence of family 2, 3 and 5 receptors in Dictyostelium was indeed surprising because they had been thought to be animal-specific. GABA (gamma amino butyric acid), the principal inhibitory neurotransmitter in mammalian brain, signals through ionotropic (GABA(A)/GABA(C)) and metabotropic GABA(B) receptor systems. The functional GABAB receptor is a heterodimer of receptor 1 and receptor 2 subtypes. The Dictyostelium genome harbours 17 different genes encoding GABAB receptor like proteins each having slightly closer resemblance to one of each subtype. Yeast two-hybrid studies led us speculate that the C termini of these receptors alone may not be sufficient for their interactions to form homo or heterodimers. The detailed analysis of one of each type of receptors namely - GrlA and GrlJ was undertaken. Both these receptors are expressed throughout the development of Dictyostelium with a marked elevation in the later developmental stages. Mutant analysis revealed both GrlA and GrlJ to be involved in post aggregation morphogenesis in Dictyostelium. Strains lacking GrlA displayed a delay in development consistent with its expression pattern and completed development 3-4 hours later than the wild type cells. Furthermore, GrlA was found to be involved in controlling sporulation events as grlA- cells were defective in the generation of the signal required for spore differentiation, SDF-2. This may be due to a decrease in the levels of TagC, a protease present on the surface of prestalk cells that cleaves AcbA (formed in prespore cells and released) to form SDF-2 and the SDF-2 receptor, DhkA. Transcriptional profiling of the grlA- (16 h) displayed downregulation in the serum response factor (srfA) induced genes, which further strengthened the role of GrlA as an important component in sporulation and spore maturation. Our data suggest that GrlJ controls events during the transition from aggregation to post aggregation. grlJ - develops precociously after aggregation stages forming thin fingers that break several times on their way until culmination and form smaller fruiting bodies with malformed spores which are less viable. However, grlJ - does not exhibit any defect in the production or response, neither to the peptides controlling sporulation such as SDF-1 or SDF-2 nor towards GABA or Glutamate like the wild type cells

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes

o-Phthalic acid, a dead-end product in one of the two pathways of phenanthrene degradation in Pseudomonas sp. strain PP2

227-232Phenanthrene is degraded via either o-phthalic acid or 1, 2-dihydroxynaphthalene in bacteria. A soil isolate Pseudomonas sp. strain PP2 degrades phenanthrene as the sole source of carbon, but failed to utilize naphthalene [Prabhu and Phale (2003) Appl Microbiol Biotechnol 61:342-351]. Analysis of the phenanthrene-grown culture spent media of this strain by gas chromatography-mass spectrometry (GC-MS) showed accumulation of o-phthalic acid. The cell-free extract prepared from this strain showed activity of 1-hydroxy-2-naphthoic acid dioxygenase (1-H-2-NADO). The extract showed conversion of 1-hydroxy-2-naphthoic acid and 2-carboxybenzaldehyde to o-phthalic acid, as analyzed by thin layer chromatography and GC-MS. However, it failed to grow or respire on o-phthalic acid. These results suggest that besides 1, 2-dihydroxynaphthalene pathway, the strain has a truncated o-phthalic acid pathway for phenanthrene metabolism and excretes o-phthalic acid as a dead-end product, indicating the co-existence of two pathways. 1-H-2-NADO, the key enzyme of o-phthalic acid pathway is inducible, has pH optima of 7.5, does not require external addition of Fe(II) as a co-factor and is completely inhibited by 1,10-phenanthroline. Absence of product formation under anaerobic condition and stoichiometric consumption of 0.82 moles of O2 per mole of product formed confirmed the dioxygenase nature of the enzyme

Conservation and Diversification of Dileucine Signal Recognition by Adaptor Protein (AP) Complex Variants*

The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as “dileucine-based,” fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals