Intracellular trafficking in Plasmodium-infected erythrocytes

The past few years have witnessed considerable progress in molecular and biochemical studies of intracellular trafficking in malaria-infected red cells. Highlights include the identification of solute channels in the vacuolar membrane and the red blood cell membrane, a tubovesicular membrane network that delivers exogenous nutrients and drugs to the parasite, and parasite gene families that mediate adherence to endothelial cells and red cells.     

Membrane potential of Plasmodium-infected erythrocytes

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.     

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.     

Transport mechanisms in Plasmodium-infected erythrocytes: lipid rafts and a tubovesicular network

The mature human erythrocyte is a simple cell that is devoid of intracellular organelles and does not show endocytic or phagocytic activity at the plasma membrane. However, following infection by Plasmodium, the erythrocyte undergoes several morphological and functional changes. Parasite-derived proteins are exported into the erythrocyte cytoplasm and to the membrane, while several proteins are localised to the parasitophorous vacuolar membrane and to the tubovesicular membranous network structures surrounding the parasite. Recent evidence indicates that multiple host proteins, independent of the type of their membrane anchor, that exist in detergent-resistant membrane (DRM) rafts or microdomains enter this apicomplexan vacuole. The internalised host components along with the parasite-encoded transmembrane protein PfEXP1 can be detected as DRM rafts in the vacuole. It appears that in Plasmodium-infected erythrocytes lipid rafts may play a role in endovacuolation and macromolecular transport.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Biosynthesis of avermectins and lipids in Streptomyces avermitilis

Abstract The gene coding for a β- d -xylosidase (E.C. 3.2.1.37) of the thermophile Caldocellum saccharolyticum was isolated previously as part of a gene cluster which has been cloned in Escherichia coli . The enzyme characteristics were determined in E. coli using toluenized cell extracts. The pH optimum was 6.5 and temperature optimum 70°C. The enzyme was stable at 60°C and the half life at 80°C was 45 minutes. The temperature optimum and the temperature stability exceed those reported for other bacterial or fungal β- d -xylosidases. The enzyme showed no other detectable xylanolytic or cellulolytic enzyme activity.     

Biosynthesis and degradation of mammalian glycosphingolipids

Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

N-epsilon-acetylation of porcine mature erythrocytes ubiquitin

Highly purified of porcine mature erythrocytes ubiquitin were obtained according to the experimental procedure reported by Jabusch and Deutsch (1983). N-epsilon-acetylation in vitro of internal lysyl residues of ubiquitin by p-nitro-phenyl-acetate at pH 8.0 was performed. The extent of acetylation of ubiquitin was determined: about 4-5 residues (4.5 residues) of N-epsilon-lysine groups of ubiquitin were acetylated. We have assigned by Edman degradation the sites of acetylation and the sites of remaining free internal N-epsilon-lysine residues in the sequence: fully acetylated: Lys-6, Lys-11 and Lys-33. Partially free N-epsilon-lysine: Lys-27 and Lys-29 and probably Lys-48 and Lys-63. 50 cycles Edman degradation were performed on porcine ubiquitin and the first 45 N-terminal residues were identified. We have partially determined that the molecular conservation of 45 amino acid sequence of ubiquitin between cattle, man and swine since the 45 amino acid sequence out of 76 residues are identical. The amino acid composition between human and porcine ubiquitin are also identical.     

Bicarbonate/chloride transport kinetics at 37°C and its relationship to membrane lipids in mammalian erythrocytes

The rate of exchange of HCO₃^? with Cl^? at 37°C in erythrocytes of ten mammalian species was studied. The rate constant increases from 7s^?1 (ox) to 16s^?1 (rat), and is inversely proportional to the body size (log₁₀) of the species. It is found that the membrane permeability in different species is positively correlated to the red cell membrane phosphatidylcholine or arachidonate content, and is negatively correlated to the sphingomyelin or linoleate content.     

Biosynthesis of membrane lipids in rat axons

Compartmented cultures of sympathetic neurons from newborn rats were employed to test the hypothesis that the lipids required for maintenance and growth of axonal membranes must be synthesized in the cell body and transported to the axons. In compartmented cultures the distal axons grow into a compartment separate from that containing the cell bodies and proximal axons, in an environment free from other contaminating cells such as glial cells and fibroblasts. There is virtually no bulk flow of culture medium or small molecules between the cell body and axonal compartments. When [methyl-3H]choline was added to the cell body-containing compartment the biosynthesis of [3H]-labeled phosphatidylcholine and sphingomyelin occurred in that compartment, with a gradual transfer of lipids (less than 5% after 16 h) into the axonal compartment. Surprisingly, addition of [methyl-3H]choline to the compartment containing only the distal axons resulted in the rapid incorporation of label into phosphatidylcholine and sphingomyelin in that compartment. Little retrograde transport of labeled phosphatidylcholine and sphingomyelin (less than 15%) into the cell body compartment occurred. Moreover, there was minimal transport of the aqueous precursors of these phospholipids (e.g., choline, phosphocholine and CDP-choline) between cell compartments. Similarly, when [3H]ethanolamine was used as a phospholipid precursor, the biosynthesis of phosphatidylethanolamine occurred in the pure axons, and approximately 10% of the phosphatidylethanolamine was converted into phosphatidylcholine. Experiments with [35S]methionine demonstrated that proteins were made in the cell bodies, but not in the axons. We conclude that axons of rat sympathetic neurons have the capacity to synthesize membrane phospholipids. Thus, a significant fraction of the phospholipids supplied to the membrane during axonal growth may be synthesized locally within the growing axon.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.