Isolation of a carotenoid-containing sub-membrane particle from the chloroplastic envelope outer membrane of pea (Pisum sativum).

Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.     

Speciation of copper in relation to its bioavailability

Abstract

Copper speciation and bioavailability for Scenedesmus quadricauda has been studied in natural waters and in synthetic culture media. Other elements were studied simultaneously. When phosphorus and nitrogen limitation were excluded by adding these elements, copper was limiting algal growth in some natural waters. In the toxic range, growth inhibition by copper was highly correlated with copper detected by electrochemical methods and with calculated free copper.

Copper was toxic to S. quadricauda when free copper concentrations roughly exceeded 10^?10.5 M, and was limiting for values somewhere lower than 10^?12.5 M. Because we found copper limitation in some natural water samples, free copper concentration in those water samples therefore must have been lower than 10^?12.5 M.

The hypothesis that the free metal concentration rather than the total concentration determines bioavailability was confirmed for copper, cobalt and zinc.     

Distinct temporospatial expression patterns of glycolysis-related proteins in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the sixth most frequent human cancer worldwide and is characterized by rapid progression as well as resistance to systemic chemotherapy. Recently, glycolysis has emerged as a potent driving force of tumor growth and therapy failure. The precise role of glycolysis for the pathogenesis of human HCC has not been elucidated thus far. Therefore, we have conducted a comprehensive analysis of the expression patterns of central glycolysis-related factors [glucose transporter-1 and -2 (Glut-1 and Glut-2), phosphoglycerate kinase-1 (PGK-1) and hypoxia-inducible factor-1α (HIF-1α)] in a large cohort of benign and malignant human liver samples. PGK-1 protein and gene expression was scant in normal liver, elevated in cirrhotic livers and most intense in HCC. Strong immunoreactivity of Glut-2 was noted in cirrhotic livers, whereas in HCC it was only expressed in 50% of examined cases. Strikingly, PGK-1 as well as Glut-2 protein expression was indicative of poor patient prognosis. Glut-1 protein was absent in neoplastic hepatocytes but prominent in tumor-associated endothelial cells. Specific nuclear staining of HIF-1α was noted in only 12% of HCC samples. Our data point toward a tumor-promoting function of glycolysis in HCC and establish PGK-1 as an independent prognostic parameter. Furthermore, the endothelial-specific expression of Glut-1 makes a special dependence of vessels on glucose reasonable to assume. In summary, we believe our analysis warrants the validation of glycolytic inhibitors as innovative treatment approaches of human HCC. Christoph Benckert and Thorsten Cramer have contributed equally to this work.     

Structural characterization of an ATPase active F1-/V1 -ATPase (alpha3beta3EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.     

Ultrastructure of the protonephridia of Seison annulatus (Rotifera)

Summary Each of the two protonephridial systems of Seison annulatus consists of three sections which are separated by cell borders with septate junctions: (a) a terminal syncytium with eight terminal organs and a capillary canal, (b) a canal syncytium which is divided into a multiciliary canal region and a main canal region, and (c) a nephroporus cell. The terminal syncytium is branched and linked twice to the canal syncytium. The supporting structure of each filtration barrier is a hollow cylinder which is perforated by pores and lacks microvilli (pillars). A protonephridial spine is situated in the multiciliary canal region and stabilizes the neck region. The ored, hollow cylinder and the protonephridial spine are new characteristics for the Rotifera.     

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

Cellulose synthase-like proteins in the D family share high levels of sequence identity with the cellulose synthase proteins and also contain the processive beta-glycosyltransferase motifs conserved among all members of the cellulose synthase superfamily. Consequently, it has been hypothesized that members of the D family function as either cellulose synthases or glycan synthases involved in the formation of matrix polysaccharides. As a prelude to understanding the function of proteins in the D family, we sought to determine where they are located in the cell. A polyclonal antibody against a peptide located at the N-terminus of the Arabidopsis D2 cellulose synthase-like protein was generated and purified. After resolving Golgi vesicles from plasma membranes using endomembrane purification techniques including two-phase partitioning and sucrose density gradient centrifugation, we used antibodies against known proteins and marker enzyme assays to characterize the various membrane preparations. The Arabidopsis cellulose synthase-like D2 protein was found mostly in a fraction that was enriched with Golgi membranes. In addition, versions of the Arabidopsis cellulose synthase-like D2 proteins tagged with a green fluorescent protein was observed to co-localize with a DsRed-tagged Golgi marker protein, the rat alpha-2,6-sialyltransferase. Therefore, we postulate that the majority of Arabidopsis cellulose synthase-like D proteins, under our experimental conditions, are likely located at the Golgi membranes. Furthermore, protease digestion of Golgi-rich vesicles revealed almost complete loss of reaction with the antibodies, even without detergent treatment of the Golgi vesicles. Therefore, the N-terminus of the Arabidopsis cellulose synthase-like D2 protein likely faces the cytosol. Combining this observation with the transmembrane domain predictions, we postulate that the large hydrophilic domain of this protein also faces the cytosol.     

Sindbis virus glycoproteins: effect of the host cell on the oligosaccharides.

Sindbis virus was grown in four different host cells and the carbohydrate portions of the glycoproteins were analyzed. Sindbis virus grown in BHK-21 cells has more sialic acid and galactose than Sindbis virus grown in chicken embryo cells. In other respects the carbohydrates from virus grown in these two hosts are very similar. Sindbis virus grown either in chick cells transformed by Rous sarcoma virus or in BHK cells transformed by polyoma virus was also examined. In comparisons of virus from normal and transformed cells, differences in the amount of sialic acid were observed; but otherwise the carbohydrate structures appeared basically similar. The growth conditions used for the host cell also affected the degree of completion of the carbohydrate chains of the viral glycoproteins.     

Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.     

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.