Vacuolar Complex Research Articles

Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease-Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock-down of endocytosis gene Rvs161 improved the concentration of α-amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock-down mutants in Rvs161 and End3 genes with 2-fold and 3-fold α-amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae. This result is likely generalizable to other eukaryotic secretion hosts.

Quel effet propre sur le taux de prothrombine de la N-acétylcystéïne administrée pour intoxication au paracétamol ?

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding γ-glutamyltranspeptidase (γGT). This is confirmed by the absence of the corresponding activity of γGT in the mutant with disrupted GGT1 gene. It was shown that γGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a γGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.

Role of γ-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.

Evolution in Caffeoylquinic Acid Content and Histolocalization During Coffea canephora Leaf Development

Caffeoylquinic acids are cinnamate conjugates derived from the phenylpropanoid pathway. They are generally involved in plant responses to biotic and abiotic stress and one of them, chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA), is an intermediate in the lignin biosynthesis pathway. Caffeoylquinic acids, and particularly 5-CQA, are accumulated in coffee beans, where they can form vacuolar complexes with caffeine. Coffea canephora beans are known to have high caffeoylquinic acid content, but little is known about the content and diversity of these compounds in other plant parts. To gain new insights into the caffeoylquinic acid metabolism of C. canephora, caffeoylquinic acid content and in situ localization were assessed in leaves at different growth stages. HPLC analyses of caffeoylquinic acid content of leaves was conducted in conjunction with detailed histochemical and microspectrofluorometrical analysis. HPLC analyses revealed that caffeoylquinic acid content was 10-fold lower in adult than in juvenile leaves. The most abundant cinnamate conjugate was 5-CQA, but dicaffeoylquinic acids (particularly in juvenile leaves) and feruloylquinic acids were also present. Using specific reagents, histochemical and microspectrofluorometrical analysis showed that caffeoylquinic acids (mono- and di-esters) were closely associated with chloroplasts in very young leaves. During leaf ageing, they were found to first accumulate intensively in specific chlorenchymatous bundle sheath cells and then in phloem sclerenchyma cells. The association with chloroplasts suggests that caffeoylquinic acids have a protective role against light damage. In older tissues, their presence in the leaf vascular system indicates that they are transported via phloem and confirms their involvement in lignification processes. In accordance with the hypothesis of a complex formation with caffeine, similar tissue distribution was observed for alkaloids and this is further discussed.

Expression, purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p).

The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F1F0-ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% alpha-helix and 32-42% beta-sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five alpha-beta sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.

The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein.

The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis, dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed.

Compartmentation of caffeine and related purine alkaloids depends exclusively on the physical chemistry of their vacuolar complex formation with chlorogenic acids

Coffea seeds accumulate purine alkaloids (PuAs) and chlorogenic acids (CGAs) in a correlated manner: a high concentration of PuAs (mostly caffeine) accompanies a considerable accumulation of CGAs (mostly 5-caffeoylquinic acid; 5-CQA) and vice versa. Since caffeine and related PuAs per se freely penetrate cell-, tissue-, and organ-related barriers, we suggested that the physico-chemically well-characterized PuA-CGA complex is the crucial mechanism of PuA sequestration in these plant species and therefore the cause of the above-mentioned correlation. Suspension-cultured coffee ( C. arabica) cells produce the complex partners in readily measurable concentrations, and were therefore an ideal system for investigating the in situ significance of complex formation. The partition of caffeine between cells and medium was studied in relation to the concentration of 5-CQA which, like the complex, is confined to the vacuole. Induction of the phenolic pathway was monitored by measuring PAL activity. To create concentration ranges of the complex partners as wide as possible, the cultures were subjected to various conditions such as the addition of a photoperiod or methyljasmonate (both stimulating secondary product formation), 2-aminoindan-2-phosphonic acid (2-AIP) (a most potent inhibitor of PAL), and exogenous caffeine. In all experimental sets, compartmentation of caffeine (and also that of theobromine) was highly correlated to the concentration of 5-CQA. In addition, inhibition of 5-CQA synthesis by 2-AIP consequently led to a reduction of caffeine biosynthesis whereas exogenous caffeine evoked the synthesis of the phenolic counterpart (5-CQA), this indicating a regulatory connection between the complex partners. When the cell cultures were transferred from 25° to 10°, or to 37°, caffeine shifted rapidly, as expected, from the medium into the cells or vice versa. Moreover, a modelling study showed that complex formation almost fully explains the measured degree of compartmentation. Similarly, in tissues and organs of the intact coffee plant the driving force of caffeine compartmentation was also shown to be defined by the physical chemistry of the complex. Finally, all caffeine-containing plants may have evolved basically one common strategy to sequester PuAs, i.e. the vacuolar allocation of high concentrations of one or several complexing phenols.

Anterograde transport of algal scales through the Golgi complex is not mediated by vesicles

The organelles of the secretory pathway are characterized by specific organization and function but they communicate in different ways with intense functional crosstalk. The best known membrane-bound transport carriers are known as protein-coated vesicles. Other traffic mechanisms, despite the intense investigations, still show incongruences. The review intends to provide a general view of the mechanisms involved in membrane traffic. We evidence that organelles’ biogenesis involves mechanisms that actively operate during the entire cell cycle and the persistent interconnections between the Endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) and endosomes, the vacuolar complex and the plasma membrane (PM) may be seen as a very dynamic membrane network in which vesicular traffic is part of a general maturation process.

The perturbation of the endomembrane network of the chlamydomonad alga Gloeomonas by the herbicide isopropyl N‐phenyl carbamate (IPC)

Abstract. The highly complex and symmetric endomembrane network of the chlamydomonad alga, Gloeomonas kupferri, can be disrupted by the herbicide, isopropyl N‐phenyl carbamate (IPC). Upon incubation with IPC for between 30 min and 1 h, curling of the dictyosomes, proliferation of small vesicles from the trans face and swelling of the peripheral vacuolar complex takes place. Longer treatments result in degeneration of dictyosome cisternae into vesicular masses and the formation of supervacuolar complexes. Enzyme cytochemistry revealed that endomembrane ‘marker’ enzymes are altered/inhibited by IPC. A second but striking I PC‐induced effect is the production of branched, fibrillar inclusions in the cell nucleus. A discussion of I PC‐induced affects on ion regulation and microtubule/MTOC distribution in the cell is presented.

Ultrastructure of Cells in the Overwintering Dormant Shoot Apex of Rhododendron maximum L.

Tissue arrangement and cell structure of terminal leaf buds of Rhododendron maximum were observed while in the dormant overwintering condition. The apical anatomy was typical of angiosperms with a two-cell-layer tunica superior to a corpus which gives rise to a poorly defined rib meristem-pith region below. The tunica-corpus cells had thin cell walls with many plasmodesmata; cells of the rib meristem pith, cortex, and procambium had thick primary cell walls with manifold pit fields. The cells of all these regions contained large ovoid or lobate nuclei, small proplastids with lipid droplets and phytoferritin, abundant dictyosomes, lipid bodies, and small vacuoles which appeared to arise from abundant myeloid figures. Tannins produced in the myeloid vacuolar complexes accumulated in the central vacuoles of cells of the rib meristem, pith, and cortex.

Rhodomonas lacustris(Pascher & Ruttner) Javornicky (Cryptomonadida): Ultrastructure of the Vegetative Cell

ABSTRACTThe ultrastructure ofRhodomonas lacustriswas examined using scanning and transmission electron microscopy, and the pigments were scanned in vivo by the opal plate technique. The periplast is composed of hexagonal plates, with trichocysts at the corners. This cryptomonad lacks a gullet but has a deep ventral furrow, in which more than 20 large ejectosomes are located. The chloroplast has mainly three thylakoids per lamella, and it is structurally reminiscent of that of certain haptophycean flagellates. The pigments appear to be as usual within the photosynthetic cryptomonads, with phycoerythrin type PE I (phycocyanin was not detected). Other cellular inclusions (e.g., lysosome‐like vesicles and the contractile vacuolar complex) are also discussed.

Micromorphological aspects of the development of rubella virus in BHK-21 cells.

Sequential effects of rubella virus infection in BHK-21 cells were studied by electron microscopy of thin sections of control and infected cells, 2 to 7 days after infection. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of virions from vacuole membranes and involvement of the endoplasmic reticulum. Newly formed endoplasmic reticulum cisternae encircled and segregated virionforming vacuoles together with other cellular elements. Large vacuolar complexes with numerous virus particles developed, and virus release from these areas occurred with disruption at the cell periphery. The viral particles, with a mean diameter of about 56 nm, consisted of an electron-dense core surrounded by a less dense capsid, enveloped by a typical unit membrane derived from the vacuole membrane.