The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.     

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.     

Lipid Accumulation, Lipid Body Formation, and Acyl Coenzyme A Oxidases of the Yeast Yarrowia lipolytica

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal β-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Δpox2 Δpox3 Δpox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.     

An evolving hierarchical family classification for glycosyltransferases

Glycosyltransferases are a ubiquitous group of enzymes that catalyse the transfer of a sugar moiety from an activated sugar donor onto saccharide or non-saccharide acceptors. Although many glycosyltransferases catalyse chemically similar reactions, presumably through transition states with substantial oxocarbenium ion character, they display remarkable diversity in their donor, acceptor and product specificity and thereby generate a potentially infinite number of glycoconjugates, oligo- and polysaccharides. We have performed a comprehensive survey of glycosyltransferase-related sequences (over 7200 to date) and present here a classification of these enzymes akin to that proposed previously for glycoside hydrolases, into a hierarchical system of families, clans, and folds. This evolving classification rationalises structural and mechanistic investigation, harnesses information from a wide variety of related enzymes to inform cell biology and overcomes recurrent problems in the functional prediction of glycosyltransferase-related open-reading frames.     

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.     

Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.     

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance

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