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PubMed1998

The "8-kD" cytoplasmic dynein light chain is required for nuclear migration and for dynein heavy chain localization in Aspergillus nidulans.

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the "8-kD" cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.

Amino Acid Sequence↗
PubMed2000

Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

Amino Acid Substitution↗
PubMed1968

Cytoplasmic label following tritiated thymidine treatment of Allium cepa L. roots. Cytochemical and electron microscope study.

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-(3)H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-(3)H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-(3)H exclusively labels DNA.

Autoradiography↗
PubMed1974

Electron microscope study of mitochondrial 60S and cytoplasmic 80S ribosomes from Locusta migratoria.

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl(2). Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of approximately 270 x 210 x 215 A; cytoplasmic ribosomes measure approximately 295 x 245 x 255 A. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is approximately 130 A, while the diameter of cytoplasmic ribosomes is approximately 180-200 A. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.

Animals↗
PubMed1981

Relation between cell activity and the distribution of cytoplasmic actin and myosin.

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

Actins↗
PubMed1992

Phagocytic chimeric receptors require both transmembrane and cytoplasmic domains from the mannose receptor.

Phagocytosis has traditionally been viewed as a specialized function of myeloid and monocytic cells. The mannose receptor (MR) is an opsonin-independent phagocytic receptor expressed on tissue macrophages. When human MR cDNA is transfected into Cos cells, these usually non-phagocytic cells express cell surface MR and bind and ingest MR ligands such as zymosan, yeast, and Pneumocystis carinii. Expression of cDNA for Fc gamma RI (CD64), the high-affinity Fc receptor, in Cos cells confers binding but barely detectable phagocytosis of antibody-opsonized erythrocytes (EA). We report here that chimeric receptors containing the ligand-binding ectodomain of the Fc receptor and the transmembrane and cytoplasmic domains of the MR ingest bound EA very efficiently, whereas chimeras with the Fc receptor ecto- and transmembrane domains and the MR tail, or the Fc receptor ecto- and cytoplasmic domains and the MR transmembrane region, are significantly less phagocytic. All of the chimeric receptors bind ligand with equal avidity, but gain of functional phagocytosis is only conferred by the MR transmembrane and cytoplasmic domains. Endocytosis of monomeric immunoglobulin G by chimeric receptors demonstrates a similar pattern, with optimal uptake by the chimera containing both tail and transmembrane regions from the MR. The chimeric receptors with only the transmembrane or the cytoplasmic domain contributed by the MR were less efficient. Site-directed mutagenesis of the single tyrosine residue in the cytoplasmic tail (which is present in a motif homologous to an endocytosis consensus motif in the LDL receptor cytoplasmic tail [Chen, W.-J., J. L. Goldstein, and M. S. Brown. 1990. J. Biol. Chem. 265:3116]) reduces the efficiency of phagocytosis and endocytosis to a similar extent.

Animals↗
PubMed1989

Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.

Animals↗
PubMed2001

The cytoplasmic domain of the integrin alpha9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner.

The integrin alpha9 subunit forms a single heterodimer, alpha9beta1. The alpha9 subunit is most closely related to the alpha4 subunit, and like alpha4 integrins, alpha9beta1 plays an important role in leukocyte migration. The alpha4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the alpha9 cytoplasmic domain has similar effects, a series of chimeric and deleted alpha9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an alpha9beta1-specific ligand. Like alpha4, the alpha9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the alpha9 cytoplasmic domain and to a similar level as alpha4. In paxillin(-/-) cells, alpha9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the alpha9-paxillin interaction prevented alpha9 from inhibiting cell spreading but had no effect on alpha9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin alpha subunits enhance migration and inhibit cell spreading are distinct and that the alpha9 and alpha4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

Amino Acid Motifs↗
PubMed1997

The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Animals↗
PubMed1997

Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.

The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.

Amino Acid Sequence↗
PubMed1998

Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in paramecium.

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the alpha- and beta- tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

Amino Acid Sequence↗
PubMed1997

erbB-2 oncoprotein expression in breast carcinoma. Poor prognosis associated with high degree of cytoplasmic positivity using CB-11 antibody.

The relationship between immunohistochemically measured membranous expression of erbB-2 oncoprotein and prognosis in breast cancer is controversial, and the relationship between cytoplasmic positivity and prognosis has been minimally investigated. The clinical course of 300 women with infiltrating breast carcinoma, whose tumors were stained for erbB-2 oncoprotein with CB-11 monoclonal antibody, was followed for 5 to 79 months (median, 41 months). Higher numbers of cancer-related deaths were observed in patients with moderate-to-strong cytoplasmic positivity (15/60, 25%) and strong membranous positivity (9/29, 31%) than in patients with negatively stained tumors (8/53, 15%) or tumors with weaker degrees of cytoplasmic or membranous positivity (rate similar to or less than negative tumors). Relapse-free survival was also significantly shorter in patients with tumors with strong membranous positivity and moderate-to-strong cytoplasmic positivity; much of the latter relationship was due to a high number of patients who had erbB-2-positive tumors and who were never disease free. A significant association between decreased relapse-free survival and moderate-to-strong cytoplasmic (but not membranous) positivity persisted even after correction for other variables associated with poor outcome (histologic grade, tumor size, lymph node involvement, and hormone receptor status). Moderate-to-strong erbB-2 oncoprotein cytoplasmic positivity, measured with CB-11 antibody, is associated with a poor prognosis and seems to have biologic significance.

Breast Neoplasms↗
PubMed1985

Natural selection with nuclear and cytoplasmic transmission. II. Tests with Drosophila from diverse populations.

Observations of intraspecific variation in organelle DNA have prompted a renewed interest in the evolutionary consequences of cytoplasmically transmitted factors. Attempts to quantify the significance of cytoplasmic effects are frequently limited by the difficulty in partitioning the cause of reciprocal cross differences among a series of possibilities. In the experiment reported here the nuclear genomes of a set of six lines of Drosophila melanogaster from diverse geographic locations were replaced in a series of cytoplasms. The segregation of the SM5 balancer chromosome was scored in a factorial design, and the data allowed a partitioning of variance such that cytoplasmic effects were distinguished from maternal effects and meiotic drive. An attempt was made to avoid the confounding problem of hybrid dysgenesis by performing the entire experiment (including chromosomal extractions) in a P cytotype. Results indicated a significant contribution of cytoplasm to the variance in SM5 segregation. Error variance showed an increasing trend as the experiment proceeded, and additional tests indicated that this was due to an accumulation of chromosomal mutations. These findings are interpreted in light of the population genetic theory that addresses the maintenance of cytoplasmic polymorphism.

Analysis of Variance↗
PubMed2000

Three leucine-rich sequences and the N-terminal region of double-stranded RNA-activated protein kinase (PKR) are responsible for its cytoplasmic localization.

The double-stranded RNA-activated-protein kinase PKR was originally identified as a ribosomal protein that regulates protein synthesis at the translational level. While PKR locates predominantly to the cytoplasm, nuclear or nucleolar species of PKR have been detected. Here, we demonstrate that PKR possesses three leucine-rich sequences resembling nuclear export signals (NESs). Enhanced green fluorescent protein (EGFP) fused to one of these sequences and transfected in COS-1 cells exhibited predominant cytoplasmic staining, which was abrogated by a leucine to alanine substitution. In addition, Leptomycin B (LMB), an inhibitor of NES-mediated nuclear export, inhibited the cytoplasmic localization of EGFP-NES, indicating the potential activity of these stretches as NESs. Although EGFP fused to a PKR with three NES mutations still located to the cytoplasm, an additional N-terminal deletion impaired the cytoplasmic predominance, suggesting that the N-terminal region is also required for localization. These results suggest that the cytoplasmic localization of PKR is regulated by NESs as well as the N-terminal sequence.

Active Transport, Cell Nucleus↗
PubMed1989

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli.

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1981

Newly synthesized L-[14C]dopamine failed to accumulate immediately in synaptic vesicles within isolated rat brain synaptosomal cytoplasm.

It was examined whether or not enzymically synthesized L-dopamine could be immediately incorporated and stored in synaptic vesicles within a synaptosomal cytoplasm fraction isolated from the rat brain. The synaptosomal cytoplasm was isotonically prepared by disrupting the synaptosomes in 0.32 M sucrose using an N2-gas pressure homogenizer. Synaptic vesicles present in the isolated synaptosomal cytoplasm appeared to be osmotically sensitive (i.e., not leaky vesicles) under iso-osmotic conditions. DOPA decarboxylase [EC 4.1.1.28] present in the soluble cytosol region synthesized L-[14C]dopamine from added L-[14C]DOPA. However, the newly synthesized L-[14C]dopamine was not immediately incorporated in the synaptic vesicles within the synaptosomal cytoplasm during the reaction period of 20 min at 30 degrees C. Plain synaptic vesicles prepared by the method of Kadota and Kadota (10) also failed to take up the newly synthesized dopamine under the same conditions. It was found that soluble protein(s) in the synaptosomal cytoplasm was able to bind as much added L-[3H]dopamine as 35 pmol per mg of protein, and the binding ability seemed to inhibit the amine uptake by synaptic vesicles within the synaptosomal cytoplasm. The [3H]dopamine bound to the protein(s) became unbound in the presence of Ca2+ (5 mM). This effect of Ca2+ may have an important role in the releasing process of neurotransmitters.

Animals↗
PubMed2001

Different cytoplasmic calcium contents among three species of Characeae.

Internodal cells of three species of Characeae, Nitella flexilis, Nitella axilliformis and Chara corallina, were analyzed for the contents of Ca(2+ )and Mg(2+) in the cytoplasm. To avoid contamination of Ca(2+) from the cell wall and vacuole, the vacuolar sap was replaced with a sorbitol solution containing Sr(2+) by the vacuolar perfusion method after the cell had been treated with Sr(2+). No significant difference in the cytoplasmic content of Mg(2+) was found among three species of Characeae, but significant differences in the cytoplasmic content of Ca(2+) were observed among them. The cytoplasmic Ca(2+) content of N. flexilis was 2.0 times that of N. axilliformis and 3.3 times that of C. corallina. The cytoplasmic drop was furthermore separated into two fractions: a chloroplast-free fraction and a chloroplast fraction. In the chloroplast-free fraction the Ca(2+) content of N. flexilis was 2.3 times that of C. corallina and 2.0 times that of N. axilliformis, while the Mg(2+) content was the same among the three species. In the chloroplast fraction N. flexilis contained about seven times more Ca(2+) and about two times more Mg(2+) than C. corallina. The difference in the cytoplasmic Ca(2+ )content was discussed in relation to the difference in the capacity for the hydration-induced Ca(2+) release existing among the three species.

Calcium↗
PubMed2003

Variant of perivitelline membrane glycoprotein ZPC of Japanese quail (Coturnix japonica) lacking its cytoplasmic tail exhibits the retention in the endoplasmic reticulum of Chinese hamster ovary (CHO-K1) cells.

Avian perivitelline membrane, an investment homologous to the mammalian zona pellucida, is composed of at least two glycoproteins. Our previous studies demonstrated that one of its components, ZPC, which is synthesized in the ovarian granulosa cells, is secreted after carboxy-terminal proteolytic processing, and this event is a prerequisite event for ZPC secretion in quail. In the present study, we examined the role of the cytoplasmic tail, which is successfully removed after proteolytic processing, in membrane transport, proteolytic processing, and the secretion of quail ZPC. In pursuit of this, we produced a truncated ZPC mutant lacking the cytoplasmic tail located in its C-terminus and examined its expression in the mammalian cell line. Western blot analyses demonstrated that the cytoplasmic tail-deficient ZPC was neither secreted nor underwent proteolytic processing in the cells. Immunofluorescence analysis and the acquisition of resistance to endoglycosidase H digestion of the cytoplasmic tail-deficient ZPC demonstrated that the deletion of the cytoplasmic tail interferes with the intracellular trafficking of the protein from the endoplasmic reticulum to the Golgi apparatus. These results indicate that the cytoplasmic tail of quail ZPC might possess the determinant responsible for the efficient transport of the newly synthesized ZPC from the endoplasmic reticulum to the Golgi apparatus.

Animals↗