Putative Golgi Research Articles

Palmitoylation of KSHV pORF55 is required for Golgi localization and efficient progeny virion production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remain elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies.

Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Cell wall integrity is compromised under temperature stress in Schizosaccharomyces pombe expressing a valproic acid-sensitive vas4 mutant

Valproic acid (VPA) is widely used as a eutherapeutic and safe anticonvulsant drug, but the mechanism is not well elucidated. Histone deacetylases (HDACs) were first identified as direct targets of VPA. Many loss-of function mutants in S. pombe have been shown to be VPA sensitive but not sensitive to other HDAC inhibitors, such as sodium butyrate or trichostatin A (TSA). This difference suggests that there are multiple VPA target genes. In the current study, we isolated a VPA-sensitive (vas) mutant, vas4-1, and cloned the VPA target gene vas4+/vrg4+ by performing complementation experiments. The vas4+/vrg4+ gene encodes a putative Golgi GDP-mannose transporter, Vrg4, which is highly homologous with ScVrg4p. Physiological experiments indicated that SpVrg4p is involved in maintaining cell wall integrity (CWI) under high- or low-temperature stress. The results of a coimmunoprecipitation assay suggested that SpVrg4p may be transferred from the ER to the Golgi through SpGot1p loaded COPII vesicles, and both single and double mutations (S263C and A271V) in SpVrg4p compromised this transfer. Our results suggested that CWI in S. pombe is compromised under temperature stress by the VPA-sensitive vas4 mutant.

Bioinformatic analysis and genetic engineering approaches for recombinant biopharmaceutical glycoproteins production in microalgae

In recent years, interest in the use of microalgae as cell factories to produce biopharmaceutical glycoproteins increased observably. Microalgae often produce biopharmaceutical glycoproteins through post-translational modification of proteins via N-glycosylation. The biophysical properties of biopharmaceutical glycoproteins such as folding, stability, and molecular recognition are influenced by the N-glycosylation process. Hence, to better understand the activity and function of biopharmaceutical glycoproteins there is a need for comprehensive knowledge on the mechanisms and pathways of N-glycosylation in microalgae. In this study, whole genome databases of different microalgae were assessed for the presence as well as the evolutionary relationship of putative Golgi glycosyltransferases (GTs) and glycosidases (GSs) using comprehensive bioinformatic techniques. The results showed that the presence and number of GTs, GSs, and mature N-glycan structures were organism specific. Since GTs and GSs had complex phylogenetic trees and conserved motif architectures, they potentially had complex evolutionary relationships and diverse functions in different organisms. Microalgal and human N-glycosylation pathways were highly distinct, and this indicated that many efforts must be made to produce humanized biopharmaceutical glycoproteins in microalgae. Hence, genetic engineering approaches were proposed for five different microalgae to remodel the N-glycosylation into human-compatible N-glycans. All in all, the bioinformatic analysis and the proposed genetic engineering in microalgae are essential for the future in producing functional pharmaceutical proteins with humanized N-glycosylation.

Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings.

The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.

Abstract A093: Interferon gamma and post-translational modifications control the dynamics and plasma membrane association of IL15RA expressed by Triple Negative Breast Cancer (TNBC)

Abstract Background: IL15RA forms part of a heterotrimeric receptor with IL2RB and IL2RG and plays a role in proliferation and differentiation of cytotoxic T-cells. IL15RA gene copy number is amplified in triple negative breast cancer leading to higher expression of an isolated IL15RA receptor component and trans-presentation of IL15 driving both an autocrine proliferative signalling cascade and activation of immune cells (Marra et al., Cancer Res. 2014 Sep 1;74(17):4908-21). GPR89/GPHR is a Golgi-complex-associated proton channel that controls Golgi complex intraluminal pH and post-translational modification of plasma membrane and secretory proteins (Maeda et al., Nat Cell Biol 10(10): 1135-1145). We have found GPR89/GPHR upregulated and overexpressed in breast cancer. Aim: To investigate the dynamics of IL15RA/IL15 in TNBC-derived cells in response to 1) inflammatory cytokines typically abundant in cancer tissue such as interferon gamma (IFN-G) and 2) upon perturbation of the putative Golgi complex associated trafficking function caused by TN-specific upregulation of GPR89/GPHR Methods: We measured the expression and plasma membrane localization of Il15RA in different TNBC-derived cell lines by flow cytometry on intact cells and by immunoblot analysis on whole cell lysates, with or without stimulation by 20 ng/ml IFN-G for 36h. The effect of GPR89/GPHR on the above mentioned phenomena was assessed by siRNA mediated GPR89/GPHR silencing or lentiviral mediated TET-ON mediated conditional expression. Results: Flow-cytometry analysis of multiple breast cancer cell lines showed a dramatic increase in the plasma membrane-associated IL15RA in response to treatment with IFN-G. The overall amount of IL15RA didn't appear to have changed by immunoblot analysis on total cell lysates, thus suggesting that IFN-G mobilises IL15RA from intracellular stores which we could identify by IF in resting cells largely colocalising with endosomal structures. The upregulation of GPR89/GPHR which we observed occurs in ca 40% of TNBC, as well as in other breast cancer subtypes, dramatically alters the postranslational modification of plasma membrane proteins in breast cancer cell lines and concomitantly reduces the proportion of IL15RA transported to the PM in response to IFN-G stimulation. These data shed light on the intracellular dynamics of IL15RA in breast cancer cell lines, suggesting an unprecedented IFN-G regulated mobilization and transpresentation of the IL15RA/IL15 complex which is sensitive to specific and yet unknown, post-translational modifications controlled by GPR89/GPHR a novel breast cancer associated Golgi-complex proton channel. Citation Format: Pierfrancesco Marra, Sumi Mathew, Steven Catchpole, Alessandra Facchetti, Andrew Tutt. Interferon gamma and post-translational modifications control the dynamics and plasma membrane association of IL15RA expressed by Triple Negative Breast Cancer (TNBC). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A093.

The Rab GTPase YPT-1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa.

Vesicle traffic involves budding, transport, tethering and fusion of vesicles with acceptor membranes. GTP-bound small Rab GTPases interact with the membrane of vesicles, promoting their association with other factors before their subsequent fusion. Filamentous fungi contain at their hyphal apex the Spitzenkörper (Spk), a multivesicular structure to which vesicles concentrate before being redirected to specific cell sites. The regulatory mechanisms ensuring the directionality of the vesicles that travel to the Spk are still unknown. Hence, we analyzed YPT-1, the Neurospora crassa homologue of Saccharomyces cerevisiae Ypt1p (Rab1), which regulates different secretory pathway events. Laser scanning confocal microscopy revealed fluorescently tagged YPT-1 at the Spk and putative Golgi cisternae. Co-expression of YPT-1 and predicted post-Golgi Rab GTPases showed YPT-1 confined to the Spk microvesicular core, while SEC-4 (Rab8) and YPT-31 (Rab11) occupied the Spk macrovesicular peripheral layer, suggesting that trafficking and organization of macro and microvesicles at the Spk are regulated by distinct Rabs. Partial colocalization of YPT-1 with USO-1 (p115) and SEC-7 indicated the additional participation of YPT-1 at early and late Golgi trafficking steps.

Null ABCA3 in humans: Large homozygous ABCA3 deletion, correlation to clinical–pathological findings

A study was undertaken to analyze the clinical presentation, pulmonary function, and pathological features in two female siblings with neonatal pulmonary surfactant metabolism dysfunction, type 3 (MIM 610921). The clinical records of the siblings were examined; the genes encoding surfactant protein B (SFTPB), surfactant protein C (SFTPC), and ATP-binding cassette transporter 3 protein (ABCA3) were analyzed with direct sequencing and Southern blotting. The infants were homozygous for a 5,983 bp deletion in ABCA3 including exons 2-5 as well as the start AUG codon and a putative Golgi exit signal motif. Dense abnormalities of lamellar bodies at electron microscopy and absence of ABCA3 at immunohistochemical staining were in agreement with the presence of two null alleles. In addition, an increased lipid synthesis suggested a compensatory mechanism. The clinical course in the two sisters was influenced by different environmental factors like the time needed for molecular confirmation, the ventilatory assistance adopted, the occurrence of infections. A less aggressive clinical approach did not improve the course of the disease; the prognosis was always poor. Development of a fast molecular test, able to detect also structural variants, is needed.

FAM20A Mutations Can Cause Enamel-Renal Syndrome (ERS)

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A −/− molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Putative Glycosyltransferases and Other Plant Golgi Apparatus Proteins Are Revealed by LOPIT Proteomics

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

Differential Selection of Golgi Proteins by COPII Sec24 Isoforms in Procyclic Trypanosoma brucei

The Sec24 subunit of the coat protein complex II (COPII) has been implicated in selecting newly synthesized cargo from the endoplasmic reticulum (ER) for delivery to the Golgi. The protozoan parasite, Trypanosoma brucei, contains two paralogs, TbSec24.1 and TbSec24.2, which were depleted using RNA interference in the insect form of the parasite. Depletion of either TbSec24.1 or TbSec24.2 resulted in growth arrest and modest inhibition of anterograde transport of the putative Golgi enzyme, TbGntB, and the secretory marker, BiPNAVRG-HA9. In contrast, depletion of TbSec24.1, but not TbSec24.2, led to reversible mislocalization of the Golgi stack proteins, TbGRASP and TbGolgin63. The latter accumulated in the ER. The localization of the COPI coatomer subunit, TbεCOP, and the trans Golgi network (TGN) protein, TbGRIP70, was largely unaffected, although the latter was preferentially lost from those Golgi that were not associated with the bilobe, a structure previously implicated in Golgi biogenesis. Together, these data suggest that TbSec24 paralogs can differentiate among proteins destined for the Golgi.

The Fukutin Transmembrane Domain: Capturing the Complexity of the Golgi Apparatus Membrane via Multiscale MD Simulations

The membrane of the Golgi apparatus is a complex mixture of lipids and proteins. In the present work we describe multiscale molecular dynamics simulations of transmembrane protein domains in model membranes that represent the in vivo Golgi environment. The transmembrane domain of glycosyltransferases is required for their correct sorting within the Golgi apparatus. The hydrophobic thickness and oligomerization state of the transmembrane domains have been proposed to mediate this sorting. Fukutin, is a putative Golgi glycosyltransferase implicated in muscular dystrophy.We employ atomistic and coarse-grained molecular dynamics simulations to investigate the stability and membrane interactions of the fukutin transmembrane domain, using various models of the membrane. Our atomistic simulations reveal that the fukutin transmembrane domain can exist as a stable α helix irrespective of the headgroup charge, fatty acid saturation or hydrophobic thickness of the lipid bilayer. Coarse-grained simulations reveal that the tilt angle of the fukutin transmembrane domain is highly variable and dependent upon its local environment; both the hydrophobic thickness and the headgroup charge of the lipid bilayer can alter the tilt angle of the protein. Lastly, we study the dynamics of the fukutin transmembrane domain in a mixed lipid bilayer whose composition closely mimics the complex lipid headgroup composition of the Golgi apparatus.

Electrophysiological Characterization of The Cerebellum in the Arterially Perfused Hindbrain and Upper Body of The Rat

In the present study, a non-pulsatile arterially perfused hindbrain and upper body rat preparation is described which is an extension of the brainstem preparation reported by Potts et al., (Brain Res Bull 53(1):59–67), 1. The modified in situ preparation allows study of cerebellar function whilst preserving the integrity of many of its interconnections with the brainstem, upper spinal cord and the peripheral nervous system of the head and forelimbs. Evoked mossy fibre, climbing fibre and parallel fibre field potentials and EMG activity elicited in forelimb biceps muscle by interpositus stimulation provided evidence that both cerebellar inputs and outputs remain operational in this preparation. Similarly, the spontaneous and evoked single unit activity of Purkinje cells, putative Golgi cells, molecular interneurones and cerebellar nuclear neurones was similar to activity patterns reported in vivo. The advantages of the preparation include the ability to record, without the complications of anaesthesia, stabile single unit activity for extended periods (3 h or more), from regions of the rat cerebellum that are difficult to access in vivo. The preparation should therefore be a useful adjunct to in vitro and in vivo studies of neural circuits underlying cerebellar contributions to movement control and motor learning.

Similarities between UDP-Glucose and Adenine Nucleotide Release in Yeast: Involvement of the Secretory Pathway

Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Delta was reduced by deletion of the YEA4 UDP- N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.

The Familial Mediterranean Fever Protein Interacts and Colocalizes with a Putative Golgi Transporter

Abstract. The biological function of pyrin, the protein mutated in Familial Mediterranean Fever (FMF), has not been elucidated. Based on sequence homology, a transcription factor activity was proposed for this neutrophil-specific protein. In a yeast two-hybrid assay, neither transcription activation activity nor any self interaction was detected for pyrin. Screening of an expression cDNA library of peripheral blood leukocytes using as bait the carboxyl portion of pyrin (amino acids 557–781), which contains most of the FMF mutations, led to the identification of P/M-IP1 (pyrin/marenostrin interacting protein 1). A splice variant of P/M-IP1, GTC-90, had previously been described as a component of the 13S hetero-oligomeric protein complex that stimulates in vitro Golgi transport. We have now shown that P/M-IP1 colocalizes with pyrin in the perinuclear cytoplasm of Cos-7 cells and that the interaction between these two proteins is impaired by FMF causing mutations in pyrin. These data suggest that, at some stage of its functional pathway, pyrin resides in the cytoplasm and might be involved in, or impacted by, cellular protein sorting by the Golgi apparatus. The data also imply that P/M-IP1 may be involved in the abnormal inflammatory response that occurs in patients with FMF.

Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located approximately 9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.

CopA:GFP localizes to putative Golgi equivalents inAspergillus nidulans

The Golgi complex is a main component of the eukaryotic secretory system and functions to modify nascent proteins sent from the endoplasmic reticulum. Ultrastructural studies of filamentous fungi have shown Golgi to be individual smooth membrane cisternae that are referred to as Golgi equivalents or dictyosomes. The Aspergillus nidulans copA gene encodes a homolog of mammalian coat protein (alpha-COP), a constituent of the Golgi-localized COPI vesicle coat. Here, the localization of A. nidulansalpha-COP was examined in live cells using the reporter green fluorescent protein (GFP). CopA:GFP localized to putative Golgi equivalents that were concentrated at hyphal tips. The localization was disrupted by the fungal metabolite brefeldin A. To investigate the significance of the microtubule cytoskeleton in the localization of putative Golgi equivalents, the copA:gfp fusion was expressed in a temperature-sensitive dynein mutant. In addition, a wild-type strain expressing copA:gfp was treated with the microtubule-disrupting drug nocodazole. The results suggest that the microtubule cytoskeleton is not the primary mechanism of localizing putative Golgi equivalents in A. nidulans.

Carnosine-like immunoreactivity in neurons of the brain of an advanced teleost, the gray mullet ( Chelon labrosus, Risso)

The distribution of the dipeptide carnosine (β-alanyl- l-histidine) and the related dipeptides anserine and homocarnosine has been studied by biochemical methods and immunohistochemistry in the brain of mammals and other tetrapods. These studies have indicated the presence of these dipeptides mainly in glial cells and in some neurons (olfactory receptor neurons and certain putative migrating neurons and neuroblasts). Here, we used immunohistochemistry with a polyclonal anti-carnosine antibody and the streptavidin–avidin method to study for the first time the distribution of carnosine/carnosine-related dipeptides in the brain of a teleost fish (the gray mullet Chelon labrosus). In order to assess the neuronal nature of carnosine-immunoreactive cells, we also used double immunofluorescence methods with antibodies to carnosine and to the neuronal protein HuC/D. The results obtained show that carnosine and/or related dipeptides are present in neurons of various brain regions. The carnosine-like-immunoreactive neuronal populations of the optic tectum and cerebellum are described in detail. In the optic tectum, immunoreactivity to carnosine/carnosine-related dipeptides is present in neurons of the stratum album and the stratum griseum centrale. In the cerebellum, immunoreactivity to these dipeptides is localized in Purkinje cells, in putative Golgi and stellate cells, and in many nerve fibers. Carnosine-like immunoreactive cells in mullet brain seem to be specific types of neurons, in line with previous results in a urodele but at variance with previous results in mammals, reptiles and anurans.

Re-defining the Golgi complex inPlasmodium falciparumusing the novel Golgi markerPfGRASP

Plasmodium falciparum, the causative agent of malaria, relies on a sophisticated protein secretion system for host cell invasion and transformation. Although the parasite displays a secretory pathway similar to those of all eukaryotic organisms, a classical Golgi apparatus has never been described. We identified and characterised the putative Golgi matrix protein PfGRASP, a homologue of the Golgi re-assembly stacking protein (GRASP) family. We show that PfGRASP is expressed as a 70 kDa protein throughout the asexual life cycle of the parasite. We generated PfGRASP-GFP-expressing transgenic parasites and showed that this protein is localised to a single, juxtanuclear compartment in ring-stage parasites. The PfGRASP compartment is distinct from the ER, restricted within the boundaries of the parasite and colocalises with the cis-Golgi marker ERD2. Correct subcellular localisation of this Golgi matrix protein depends on a cross-species conserved functional myristoylation motif and is insensitive to Brefeldin A. Taken together our results define the Golgi apparatus in Plasmodium and depict the morphological organisation of the organelle throughout the asexual life cycle of the parasite.