Showing papers on "Cellular compartment published in 1991"

Bovine papillomavirus E5 oncoprotein binds to the 16K component of vacuolar H(+)-ATPases.

Abstract: The major transforming protein of bovine papillomavirus type 1, E5, is mainly associated with endomembranes, specifically binding to a cellular protein of relative molecular mass 16,000 (16K). At the same time as transformation, E5 causes the phosphorylation of tyrosine residues in epidermal and platelet-derived growth factor receptors. We show here that the 16K protein associated with E5 is the 16K component of vacuolar ATPases. This protein is known to be an integral membrane protein in endosomes, bovine chromaffin granules, synaptic vesicles, fungal and plant vacuoles and clathrin-coated vesicles, as well as a component of gap-junction-like membrane complexes. Because proton pumps are critical for the function of cellular compartments that process growth-factor receptors, the interaction of E5 with the 16K protein could explain the pleiomorphic features of cells transformed by E5.

Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments.

Abstract: The structural proteins of the retroviral capsid are translated as a polyprotein (the Gag precursor) that is cleaved by a virally encoded protease. Processing of the human immunodeficiency virus type 1 Gag precursor Pr55 was analyzed through a combination of pulse-chase labeling, cell fractionation, and immunoprecipitation. We observed a membrane-associated processing pathway for the Gag precursor that gives rise to virions. In addition, we found that a significant amount of processing occurs in the cytoplasm of infected cells resulting in the intracellular accumulation of appropriately processed viral proteins. This observation suggests the viral protease is active in the cytoplasmic compartment of the cell. Processing of the Gag protein was blocked in both compartments by the addition of a viral protease inhibitor. A comparison of the amount of cytoplasmic processing seen in lytically infected cells with that seen in chronically infected cells showed that cytoplasmic processing was associated with the lytic infection. These observations raise the possibility that activation of the human immunodeficiency virus type 1 protease in the cytoplasm of lytically infected cells might result in the cleavage of cellular proteins and thus contribute to cytotoxicity.

Regulation of intracellular pH in plant cells

Abstract: Publisher Summary This chapter reviews the basic principles of the techniques used for intracellular pH measurement in the main cell compartments—namely, cytoplasm and vacuole Alongwith being both substrate and product in numerous metabolic reactions, protons fulfill the regulatory role of coordinating the activities of enzyme-catalyzed pathways, membrane transport, and other regulators The protons connect cellular compartments and also play important roles in intercellular traffic The sudden pH shifts may impose critical loads on the cells The technical improvements that have been brought about since the previously published reviews are described in the chapter The technique involves the extraction of cell sap and the measurement of its pH with a glass electrode It is used for various types of plant materials The principle of this technique is based on three requirements: the probe molecule is metabolically inert, only the uncharged form is membrane permeant, and the probe is not to change the pH of the respective compartment The distribution of protons within a plant cell appears as a critical element of cell organization and function

MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA.

Abstract: MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.

Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply.

Abstract: The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H(+)-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of delta pH. The maintenance of the delta pH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process.(ABSTRACT TRUNCATED AT 250 WORDS)

Trans‐Golgi reticulum

Abstract: The trans-Golgi apparatus reticulum is that portion of the Golgi apparatus located in the trans-most aspect of the stack exhibiting certain characteristic morphological and functional characteristics. The membranes of the trans-Golgi reticulum are reticular in form, thickened with plasma membrane-like characteristics and with a considerable portion of their surface covered by clathrin coats. The enzymes thiamine pyrophosphatase and sialyl- and galactosyl transferases are functional markers. Correlative studies show the trans-Golgi apparatus reticulum to be involved in glycoprotein, enzyme and receptor processing and sorting along multiple pathways. Sorting and transfer of constituents to lysosomes, to secretory granules, or to the plasma membrane emerge as dominant functions.

Nuclear calcium gradients in cultured rat hepatocytes

Abstract: Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.

Cell-free transfer and sorting of membrane lipids in spinach : donor and acceptor specificity

Abstract: The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [14C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.

Subcellular Distribution of Monoclonal Antibody Defined Epitopes on Immunodominant Mycobacterium tuberculosis Proteins in the 30‐kDa Region: Identification and Localization of 29/33‐kDa Doublet Proteins on Mycobacterial Cell Wall

Abstract: Two different groups of monoclonal antibodies (MAbs) directed to different epitopes on 30-kDa region proteins of Mycobacterium tuberculosis were isolated; MAbs 5F9, 5D5 and 5D2 reacted with a single 33-kDa protein band, whereas MAb 3A8 reacted with a distinct 29/33-kDa doublet when analysed by immunoblotting. The present paper describes the distribution of MAbs defined epitopes in the 29-33-kDa region proteins in well-characterized subcellular fractions: cytosol, plasma membrane, cell wall as well as culture filtrate of M. tuberculosis. MAbs 5F9, 5D5 and 5D2 reactive epitopes were found in cytosol, whereas 3A8 epitope is distributed in all cellular compartments of the mycobacterium as well as in the culture filtrate. Localization of these epitopes by indirect immunofluorescence and immunogold-labelling demonstrated that only 3A8 epitope is present on the cell surface of the mycobacterium. Both immunoblotting and ELISA showed that only MAb 3A8, and not 5F9, 5D5 and 5D2, reacted with secreted BCG 85 antigen complex of Mycobacterium bovis BCG. Furthermore, using an MAb 3A8-coupled affinity column, we purified antigen 3A8 from the cytosol fraction of M. tuberculosis. All these MAbs reacted with antigen 3A8 with varying degrees of intensity, thus suggesting that they are directed to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and to a single protein. Absence of 5F9, 5D5 and 5D2 epitopes in the cell wall, culture filtrate and BCG-85 complex suggests that these epitopes might have been lost during the processing of the same 33-kDa protein on its way out from cytosol to the cell wall or when the protein is secreted out into the culture filtrate. Our results demonstrate, for the first time, direct evidence of the presence of a 30-kDa region protein not only in secreted antigens but also in the cell wall and on the cell surface of the mycobacterium.

Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis

Abstract: Immunocytochemistry was used to assess the location of xanthine dehydrogenase (EC 1.1.1.204) in the infected region of nodules of cowpea (Vigna unguiculata [L.] Walpers cv. Queen Anne Blackeye). Polyclonal antibodies raised against purified cowpea xanthine dehydrogenase were used to localize this enzyme at the electron microscopic level. Sparse nonspecific labeling was observed after treatment of nodule sections with preimmune serum. Although immune serum cross-reacted with the ground cytoplasm of both infected and uninfected cells, significantly more labeling was observed in the uninfected cells. No labeling above background was observed in peroxisomes, mitochondria, proplastids, endoplasmic reticulum, cytoplasmic or peribacteroid membranes, peribacteroid spaces, or bacteroids. The enzyme is soluble and not present in any organelle or membrane. The greater concentration of xanthine dehydrogenase in the uninfected cells suggests that xanthine or a precursor to xanthine, rather than uric acid, is the intermediate that moves from infected to uninfected cells during ureide biogenesis.

Increased endoplasmic reticulum content of Phanerochaete chrysosporium INA-12 by inositol phospholipid precursor in relation to peroxidase excretion

Abstract: Haem protein excretion (i.e., lignin and manganese-dependent peroxidases) by Phanerochaete chrysosporium INA-12 was improved in response to an exogenous supply of phospholipid components (inositol and linoleic acid) as well as phosphatidylinositol (PI). Maximal enzyme productions were 46.3 and 21.1 nkat · ml−1, respectively, in inositol cultures after 3 days incubation. Cellular compartment determination by marker enzymes revealed that the enhancement of protein excretion with inositol was correlated with a proliferation of endoplasmic reticulum; cytosol and mitochondrial activity did not change. In contrast, in the presence of linoleic acid and PI total cellular activity was increased. In culture containing inositol, the intracellular phospholipid composition of strain INA-12 mycelium exhibited a two fold enrichment in PI at the expense of phosphatidylserine and its derives, phosphatidylethanolamine and phosphatidylcholine.

The Dynamic Parameter

Abstract: The viability of organisms is dependent on the controlled flow of information and metabolic/synthetic precursors between cellular compartments. Such processes are elaborated upon as a hierarchy of interdependence established between cells and tissues. Through the ebb and flow of signaling and metabolic molecules, dynamic linkages may be maintained between cells for the coordination, synchronization, and initiation of cellular cycles (Fig. 1). In this manner, organismal response to the environment may be viewed as the result of a linked web of dissipative molecular gradients across biological membranes that initiate and transmit environmental information and cellular status. Integration of these gradients over large numbers of cells and tissues collectively leads to spatial and/or temporal responses. The biological structures that serve as controllable elements for transmembrane molecular flow are generally classified as channels or pores that serve either as passive transport routes for low-molecular-weight molecules (Loewenstein, 1979; Nikaido and Nakae, 1979; Gunning and Overall, 1983) or as ion pumps or transporters requiring some type of coupled gradient dissipation or energy Open image in new window Figure 1 Dynamic linkages between organelles and cells. Chemical gradients are utilized to transmit information between the cell and the environment. The pathways involved in this transmission system are: lateral mobility of membrane receptors (1); transplasma membrane transport through channels, pores, and transporters (2); homotypic intercellular communication through gap junctions or plasmodesmata (3); nucleocytoplasmic transport (4); translysosomal or vacuolar membrane transport of H+ and ions (5); Golgi-mediated processing, secretion, and recycling (6); Golgi transport of newly synthesized proteins (cis-medial-trans) (7); heterotypic intercellular communication (8). R, N, and G represent membrane receptors, the nucleus, and Golgi, respectively. source for molecular transposition (Mitchell, 1979; Noma, 1983; Reuter et al., 1983). In most instances, the control of these channels is mediated by ligand-specific receptors that couple to the channels under activating conditions, initiating a cascade of enzymatic changes resulting in a modification of channel transport properties (Koshland, 1981; Bean et al., 1983; Hondeghem and Katzung, 1984). In other cases, transport channels and receptors are intimately linked, forming a common structure, as in the case of the nicotinic acid receptor/channel (Conti-Tronconi and Raftery, 1982).

Posttranslational Modification by Phosphorylation: Control of Cytochrome P450 and Associated Enzymes

Abstract: Regulation of proteins by posttranslational modification is of crucial importance for eucaryotic cells in particular because of the relatively long half-lives of their mRNAs and the resulting lack of rapid protein regulation at the nucleic acid level. The phosphorylation of proteins (and their dephosphorylation) has emerged as the most widespread and important posttranslational regulatory device. It is now known to occur in all types of cells and in virtually all cellular compartments and organelles (1.2).