Alternatively, the role of the glycosomal compartmentalization of em Lm /em DAT is to sequester its product, 1-acyl-DHAP, in this organelle for conversion into 1-alkyl-DHAP by the glycosomal alkyl DHAP synthase ADS1 rather than being metabolized to 1-acyl-G3P by the cytosolic alkyl/acyl-DHAP reductase for the synthesis of ester glycerolipids [2], [17]

Alternatively, the role of the glycosomal compartmentalization of em Lm /em DAT is to sequester its product, 1-acyl-DHAP, in this organelle for conversion into 1-alkyl-DHAP by the glycosomal alkyl DHAP synthase ADS1 rather than being metabolized to 1-acyl-G3P by the cytosolic alkyl/acyl-DHAP reductase for the synthesis of ester glycerolipids [2], [17]. a local self-healing skin lesion and affects approximately 1C1. 5 million patients every year [1]. Ninety percent of cases of cutaneous leishmaniasis are found in Afghanistan, Pakistan, Syria, Saudi Arabia, Algeria, Iran, Brazil, and Peru [1]. Ether glycerolipids are major components of membranes, representing approximately 20% of total cellular lipids [2], [3]. In an ether lysophosphatidylinositol anchor [11] The salient feature of LPG is the conserved domain name consisting of the Gal1,4Man1-PO4 backbone of repeat models that in are branched with galactose and arabinose residues [6], [9], [12], [13], [14]. In and related parasites [2], [16], [19], while the acyl/alkyl-DHAP reductase is usually associated with the glycosomes but its active site faces the cytoplasm [17]. Open in a separate window Physique 1 Glycerolipid biosynthetic pathways in are unknown. Friedlin V1 strain (MHOM/IL/80/Friedlin) were propagated in liquid and semi-solid M199-derived medium [2]. The null mutant and complemented collection were explained in [4]. Transfection was performed according to Ngo and colleagues [20] and selection was applied as appropriate in the presence of 20C40 g/ml G418 or 25C50 g/ml of hygromycin. To follow parasite proliferation, mid log phase parasites were diluted to 5105/ml and enumerated with a hemacytometer as a function of time. Plasmids Deletion constructs of were produced by polymerase chain reaction (PCR) using pL-BSD.LmDAT [16] as a template, and the primer pairs O33 (5-CCGGGATCCCATATGAGCTTCCCACCACCTCGG-3) and O116 (5- CGGGATCCTCACATCTTGGACAGAAGACGCTTTGCCCG-3), O41 (5-CGGGATCCTCACATCTTGGATGGCTGTGTT-3) and O111 (5-CGGGATCCATGCCCTATCACCAGTGTG-3), and O41 and O136 (5-CGGGATCCATGACGGCGAACGGCTGGC-3). The producing amplified DNA fragments were digested with to give the strains and and FV1 protein extracts were prepared as explained previously [16], [18]. Protein concentration was determined by the bicinchoninic acid assay using bovine serum albumin as a standard. DHAPAT activity was assessed by measuring the acylation rate of DHAP produced by catabolism of fructose-1,6-biphosphate by the action of an aldolase and a triose phosphate isomerase, based on a protocol established by Bates and Saggerson as explained in [16]. The specificity of [U-14C]D-fructose-1,6-biphosphate (MP Biomedicals) was 295 mCi/mmol. Digitonin fractionation and electrophoresis For digitonin treatment new end-log cells were harvested, washed once in phosphate buffered IL1 saline (PBS), and resuspended in 20 mM TrisHCl (pH 8.0), 1 mM EDTA, 1 mM DTT containing a protease inhibitor cocktail (Roche) at a cell density of 2108/ml. Aliquots of 100 l were made and supplemented with increasing (0 to 0.6 mg/ml) concentrations of digitonin (stock solution of 15 mg/ml in PBS) and incubated at 26C for 10 min. Cells were then centrifuged at Pepstatin A 20,800 g for 2 min. Supernatants were immediately removed and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses in the presence of monoclonal WIC79.3 (specific to -galactose side-chains; nice gift from S. Turco; [21]) and V5 (Invitrogen) antibodies, and polyclonal immunoglobulins specific to hypoxanthine guanine phosphoribosyltransferase (nice gift from A. Jardim; [22]), phosphomannomutase (nice gift from L. Kedzierski; [23]), and arginase (nice gift from B. Ullman; [24]) were carried out as explained previously [2], [4]. Immunofluorescence assay Immunofluorescence assay was performed with wild-type parasites expressing pXG.HV-LmDAT-C3 as described previously [16]. The recombinant His6-V5 (HV) tagged HV-and shows that orthologs of parasites of the trypanosomatidae family bear a very large N-terminal extension of approximately 650 amino acids that is absent in higher eukaryotic orthologs ([16]; Fig. 2A; data not shown). Curiously, this domain name fails to exhibit any similarity to known proteins and thus, is usually parasite specific. The function of this N-terminal extension was first investigated by creating truncated background [16]. Open in a separate window Physique Pepstatin A 2 Characterization of mutant forms of mutants lacking ether lipids synthesize slow migrating forms of LPG as a result of hyperglycosylation of its disaccharide domain name [2], [4]. Western blot analysis showed that HV-mutant, transporting a genetic deletion of the alkyl DHAP synthase gene and lacking ether lipids, does not exhibit any growth phenotype, suggesting that ether lipids are dispensable for growth and survival during the stationary phase [2]. Thus, and FV1 were fractionated in the presence of digitonin as explained in Materials and Methods. Cell supernatants were then subjected to Western blot Pepstatin A analysis in the presence of monoclonal anti-V5 antibodies (V5), and of polyclonal immunoglobulins specific to arginase (ARG) and phosphomannomutase (PMM). Equivalent of 107 cell supernants were loaded in each lane. The apparent molecular excess weight markers are shown. DHAPAT assays showed that HV-the cytosol to the endoplasmic reticulum and glycosomes where lipid biosynthesis occurs [16], [32], [33], [34]. Alternatively, the role of the glycosomal compartmentalization of em Lm /em DAT is usually to sequester its product, 1-acyl-DHAP, in this organelle for conversion into 1-alkyl-DHAP by the glycosomal alkyl DHAP synthase ADS1 rather than being metabolized to 1-acyl-G3P by the cytosolic.