Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists

During the last 4 years there has been an enormous interest in the question how iron-sulphur ([Fe-S]) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes. The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution. In yeast and probably all other eukaryotes the mitochondrion is the place where [Fe-S] clusters are synthesised, even for extramitochondrial [Fe-S] cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway. However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this. In this article I will summarise the current view of [Fe-S] cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.     

Buckwheat accumulates aluminum in leaves but not in seeds

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi) is highly resistant to Al stress and is known to be an Al-accumulator. Pot experiments were carried out in a greenhouse to investigate the accumulation of Al in leaves and seeds of buckwheat. Plants were grown for 12 weeks in a strong acid soil amended with or without CaCO₃ at a rate of 1 g kg⁻¹ soil. Old leaves accumulated as much as 10 g kg⁻¹ Al of dry weight when the plants were grown in the acid soil, while the Al concentrations in leaves immediately adjacent to seeds, seed coats, and embryos were, on average, 4516, 41.2 and 7.7 mg kg⁻¹, respectively. The Al concentration significantly decreased in leaves when the plants were grown in the limed soil, and the Al concentrations in leaves immediately adjacent to seeds, seed coats, and embryos were, on average, 1586, 21.3 and 3.1 mg kg⁻¹, respectively. These results show that seeds accumulate much less Al than buckwheat leaves. The underlying mechanisms are discussed. Section Editor: H. Lambers     

The Golgi apparatus in parasitic protists

This review summarizes the current reports on the Golgi apparatus of parasitic protists. Numerous recent publications have demonstrated that studies on intracellular traffic in parasites essentially advanced our knowledge on the Golgi structure and function, which has been traditionally based on research on yeast and mammalian cultured cells. It has been reported that the parasitic lifestyle determines the functional and structural peculiarities of the secretory systems in unrelated groups of unicellular parasites that make them different from those in mammalian and yeast cells. This review covers the best-studied protists, predominantly those of high medical importance, belonging to the following taxa: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma, Plasmodium), and Kinetoplastida (Trypanosoma, Leishmania). The morphology of the Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of the six highest taxa of Eukaryota (Adl et al., 2005) a minimum of eight groups are represented by species lacking Golgi dictiosomes. However, biochemical and/or molecular (genomic) evidence indicate that an organelle with the functions of the Golgi was present in every lineage of eukaryotes studied thus far. Loss of the Golgi organelle is a secondary event as proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. The loss might have occurred independently several times in evolution. Neither the number of stacks, nor the size of the organelle correlates with the intensity of secretion or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Golgi apparatus in parasitic protists (review of the literature)

This review summarizes modem data on Golgi apparatus of parasitic protists and demonstrates how the parasitic lifestyle determines functional and structural peculiarities of secretory systems in unrelated groups of unicellular parasites, in comparison to ones of "model systems", mammalian and yeast cells. The review covers the most well-studied protists, predominantly of high medical importance, belonging to following taxons: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma and Plasmodium), and Kinetoplastida (Trypanosoma and Leishmania). Numerous recent publications demonstrated that studies on intracellular traffic in the mentioned above parasites essentially advanced our knowledge of Golgi function, traditionally based on research of cultured mammalian and yeast cells. Morphology of Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of total six the highest taxons of Eukatyota (Adl et al., 2005) there exist at minimum eight groups represented by species lacking Golgi dictiosomes. However, biochemical and (or) molecular (genomic) evidences indicate that the organelle with functions of Golgi was present in every studied so far lineage of eukaryotes. Loss of Golgi organelle is a secondary event, which has been proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. This loss might have occurred independently several times in the course of evolution. Neither the number of stacks, nor the size of the organelle correlates with intensity of secretion, or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.     

Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships

Sequences of putative fructose-1,6-bisphospate aldolases (FBA) in five amitochondriate unicellular eukaryotes, the diplomonads Giardia intestinalis (published earlier) and Spironucleus barkhanus, the pelobiont Mastigamoeba balamuthi,the entamoebid Entamoeba histolytica, and the parabasalid Trichomonas vaginalis all belong to Class II of FBAs and are highly similar to each other (>48% amino acid identity). The five protist sequences, however, do not form a monophyletic group. Diplomonad FBAs share a most recent common ancestor, while FBAs of the three other protist species are part of a lineage that also includes sequences from a few eubacteria (Clostridium difficile, Treponema pallidum, Chlorobium tepidum). Both clades are part of the Type B of Class II aldolases, a complex that contains at least three additional lineages (subgroups) of enzymes. Type B enzymes are distant from Type A Class II aldolases, which consists of a number of bacterial and fungal enzymes and also contains the cytosolic FBA of Euglena gracilis. Class II aldolases are not homologous to Class I enzymes, to which animal and plant enzymes belong. The results indicate that amitochondriate protists acquired their FBAs from separate and different sources, involving lateral gene transfer from eubacteria, than did all other eukaryotes studied so far and underscore the complex composition of the glycolytic machinery in unicellular eukaryotes.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

Hemopexin is synthesized in peripheral nerves but not in central nervous system and accumulates after axotomy.

In adult mammals, injured axons regrow over long distances in peripheral nerves but fail to do so in the central nervous system. Analysis of molecular components of tissue environments that allow axonal regrowth revealed a dramatic increase in the level of hemopexin, a heme-transporting protein, in long-term axotomized peripheral nerve. In contrast, hemopexin did not accumulate in lesioned optic nerve. Sciatic nerve and skeletal muscle, but not brain, were shown to be sites of synthesis of hemopexin. Thus, hemopexin expression, which can no longer be considered to be liver-specific, correlates with tissular permissivity for axonal regeneration.     

Plant defensin AhPDF1.1 is not secreted in leaves but it accumulates in intracellular compartments

? Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. ? Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. ? All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. ? These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level.     

Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis

Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.     

Medial Golgi but not late Golgi glycosyltransferases exist as high molecular weight complexes. Role of luminal domain in complex formation and localization

To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, beta1,4-galactosyltransferase and alpha1, 2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to beta1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.     

Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments

Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transport. The requirement for novel components at different stages of the secretory pathway may reflect the recently recognized differences in protein transport between the Golgi stacks as opposed to the selective sorting and concentration of protein during export from the ER.     

Parental adenovirus DNA accumulates in nucleosome-like structures in infected cells.

Micrococcal-nuclease digestion of adenovirus 2(ad 2) infected HeLa cell nuclei early after infection has been used to investigate the nucleoprotein nature of parental viral DNA. Viral DNA is more susceptible to nuclease digestion than cellular DNA. The pattern of digestion products changes as digestion proceeds from an indistinct pattern 1 hour post infection(pi) to a nucleosome-like pattern at 6 hours pi. The major differences between viral and cellular nucleoprotein products were i) a subnucleosome fraction from viral DNA and ii) the repeat size of DNA in viral nucleosomes was 165 base pairs and in cellular nucleosomes, 195 base pairs. Up to 50% viral DNA in nuclei 6 hours pi seems to be in nucleosome-like structures. Such patterns are not seen on digestion of partially-uncoated virus or isolated cores.     

Golgi sulfation of the oligosaccharide chain of P0 occurs in the presence of myelin assembly but not in its absence.

To decipher the intracellular targeting mechanism by which the major glycoprotein of peripheral nerve myelin, P0, is delivered to myelin after crush injury, as well as to the lysosome after permanent transection injury of the sciatic nerve--experimental paradigms characterized by the presence and absence of axonal regeneration and subsequent myelin assembly, respectively--the role of sulfation of P0 was investigated. P0 sulfation is shown to occur within the Golgi apparatus as a post-translational modification of the oligosaccharide chain which is dependent on processing beyond the action of mannosidase I. It is associated with myelination as observed during development and after crush injury, but does not occur after transection injury, even in the presence of the mannosidase II inhibitor, swainsonine, or the lysosomotrophic agent, L-methionine methyl ester. Although P0 accumulation can be demonstrated with both agents when other precursors are used (e.g. fucose, mannose, amino acids) and indicates lysosomal targeting and delivery of P0 after the action of GlcNAc transferase I, the absence of P0 sulfation after transection suggests that the lack of this modification may result in a default mechanism for lysosomal targeting after nerve transection. Lysosomal degradation of P0 was evaluated after crush injury by pulse-chase analyses with 35SO4 and [3H] mannose in the presence and absence of chlorate, an inhibitor of ATP-sulfarylase. Although P0 sulfation of the oligosaccharide chain is a stable modification whose labeling is dramatically inhibited by chlorate, no decrease in mannose-labeled P0 was seen with chlorate even with prolonged chase times. Because of this lack of degradation of mannose-labeled P0 in the presence of chlorate in the crushed nerve, it is concluded that the absence of P0 sulfation does not result in a default mechanism for lysosomal delivery.     

Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

Puromycin does not inactivate the galactrosyltransferase of Golgi membranes.

The effects of puromycin on galactosyltransferase from four sources, rat liver and lactating sheep mammary Golgi membranes, bovine colostrum and human serum have been investigated. We do not find that the synthesis of N-acetyllactosamine is much inhibited, even in the presence of 3mM puromycin. This is in contrast to previously reported results for rat liver Golgi membranes. We interpret the low level of inhibition observed in terms of pH effects.     

ZnT7, a novel mammalian zinc transporter,accumulates zinc in the Golgi apparatus

ZnT7, a novel member of the zinc transporter (ZnT) family, was identified by searching the expressed sequence tag (EST) databases with the amino acid sequence of ZnT1. Like the other ZnT proteins, the protein (387 amino acids) predicted from this gene contains six transmembrane domains and a histidine-rich loop between transmembrane domains IV and V. We show that Znt7 is widely transcribed in mouse tissues with abundant expression in the liver and small intestine and moderate expression in the kidney, spleen, brain, and lung. An affinity-purified antibody raised against the amino acids 299-315 of mouse ZnT7 specifically reacted with the proteins with apparent molecular masses of 85, 43, and 65 kDa in small intestine and lung tissues by Western blot analysis. Immunofluorescence microscope analysis reveals that ZnT7 is localized in the Golgi apparatus and cytoplasmic vesicles. Exposure of the ZnT7-expressing Chinese hamster ovary (CHO) cells to zinc causes an accumulation of zinc in the Golgi apparatus, suggesting that ZnT7 facilitates zinc transport from the cytoplasm into the Golgi apparatus.     

Pbx raises the DNA binding specificity but not the selectivity of antennapedia Hox proteins.

We have used a binding site selection strategy to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site, TGATTGATTTGAT. An inhibitory domain located N terminal of the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. The affinities of these proteins as heterodimeric partners with Pbx1 for the selected optimal binding site are similar. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx.     

Repair of alkylated DNA: Drosophila have DNA methyltransferases but not DNA glycosylases

DNA methyltransferase activity has been identified in crude extracts of Drosophila melanogaster pupae for the removal of methyl groups from O-6 methylguanine appearing in alkylated DNA. Additionally, N-7 methylguanine and 3 methyladenine appear to be uniquely susceptible to methyltransferase activity that resides in Drosophila pupae. Consistent with this, tests to detect DNA glycosylase activity for the repair of the latter two modified bases was unsuccessful, even though a substantial loss of methyl groups from these bases was observed. Conversely, the repair of methylated purines was not detected in extracts of Drosophila embryos. The removal of methyl groups from methylated purines was dependent upon incubation temperature and was proportional to the amount of protein added to reaction mixtures. Results indicate that the methyl group is attached to protein during the repair of methylated DNA, suggesting that it is similar to the O6-methylguanine-DNA methyltransferase identified in other organisms. Although other explanations are possible, the inability to detect DNA glycosylase activity suggests that Drosophila may not rely on base excision repair for the removal of modified or nonconventional basis in DNA.