Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves.     

Two-intermediate model to characterize the structure of fast-folding proteins

This paper introduces a new model that enables researchers to conduct protein folding simulations. A two-step in silico process is used in the course of structural analysis of a set of fast-folding proteins. The model assumes an early stage (ES) that depends solely on the backbone conformation, as described by its geometrical properties—specifically, by the V-angle between two sequential peptide bond planes (which determines the radius of curvature, also called R-radius, according to a second-degree polynomial form). The agreement between the structure under consideration and the assumed model is measured in terms of the magnitude of dispersion of both parameters with respect to idealized values. The second step, called late-stage folding (LS), is based on the “fuzzy oil drop” model, which involves an external hydrophobic force field described by a three-dimensional Gauss function. The degree of conformance between the structure under consideration and its idealized model is expressed quantitatively by means of the Kullback-Leibler entropy, which is a measure of disparity between the observed and expected hydrophobicity distributions. A set of proteins, representative of the fast-folding group - specifically, cold shock proteins - is shown to agree with the proposed model.     

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

E xcretion of c ytoplasmic p roteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG‐binding protein, which is secreted via the Sec‐pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan‐binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non‐excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.     

Aggregates of oxidized proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins

Cellular senescence may be accompanied by accumulation of large aggregates of oxidized proteins, also known as lipofuscin. The hypothesis that cellular accumulation of lipofuscin-like materials (LIP) results in cell death as a result of proteasome inhibition was examined. Rat neonatal cardiomyocytes were incubated with synthetic LIP for up to 48 h. This was accompanied by increases in cellular autofluorescence (207% by 48 h; p < 0.05) and electron microscopic evidence of internalization of LIP particles. LIP incubation resulted in loss of viability (-46% by 48 h; p < 0.05) through apoptotic cell death. Although 20S-proteasome activity was increased by 74% after 6 h, both 20S- and 26S-proteasome activities were decreased after 48 h of incubation (-54% (p < 0.05) and -50%, respectively), accompanied by large increases in ubiquitinated proteins. Several proteasome-regulated proapoptotic proteins, including c-Jun (2.9-fold; p < 0.05), Bax (1.8-fold; p < 0.05), and p27(kip1) (3.2-fold; p < 0.05), were observed to be increased by 48 h. Observation of ubiquitinated homologues of Bax and p27(kip1) suggested that part of the increase was due to decreased proteasomal degradation of these proteins. The results of this study are consistent with the conclusion that accumulation of LIP results in inhibition of the proteasome, which initiates an apoptotic cascade as a result of dysregulation of several proapoptotic proteins.     

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.     

Transfer of cytoplasmic and nuclear proteins between cells in culture

Evidence is presented for transfer of proteins between cells in culture, using techniques which previously have shown RNA transfer and the lack of DNA transfer between cells in culture. These techniques involved making donor cells heavier than recipient cells by having them ingest tantalum particles. After coculture of donor and recipient cells the two cell types were separated by centri- fugation on Ficoll gradients and the recipient cells analyzed for radioactively labeled proteins that may have passed from the prelabeled donor cells.These techniques also provided evidence for passage of donor cell proteins to recipient cell nuclei. Examination of the nuclear proteins in the recipient cells revealed that histones were transferred intercellularly to a greater extent than other nuclear proteins. The histone subfractions in the recipient cell nuclei were studied by acrylamide gel electrophoresis. No major differences were found in the proportion of each histone subfraction that was transferred to the recipient cell nuclei.     

Suppression of DNA synthesis in normal, but not neoplastic, nuclei by cytoplasmic extracts from resting cells

We have previously shown that a heat-stable protein in cytoplasmic extracts from human quiescent peripheral blood lymphocytes (PBL) is capable of inhibiting the induction of DNA synthesis in isolated resting nuclei. We now report that these cytoplasmic extracts are also capable of suppressing DNA synthetic activity in replicative nuclei isolated from mitogen-activated PBL. PBL extracts had little or no inhibitory effect, however, on replicative nuclei derived from several transformed lymphoblastoid cell lines. These results suggest that the growth of normal lymphocytes may be negatively controlled by cytoplasmic inhibitory factors. Furthermore, the relative resistance of tumor cell nuclei to these inhibitory signals provides a possible explanation for the loss of growth control in neoplastic cells.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline     

Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells

Mutant cells of the HD33 subline of the Ehrlich-Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high-molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 X 10(9)) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of beta-type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.     

Intracisternal polycylinders: A cytoplasmic structure in cells of the terrestrial slug Arion empiricorum férussac (Pulmonata,Stylommatophora)

Study of the digestive organs of the slug Arion empiricorum with the electron microscopy has revealed cytoplasmic structures that we call intracisternal polycylinders (ICPC). They consist of cylinders of cytoplasm (about 550 A in diameter) arranged in sheafs within cisterns of the endoplasmic reticulum. They appear in different cell types, being most common in the digestive epithelium of the midgut. Their morphology and apparent association with other cytoplasmic organelles such as mitochondria, peroxisomes, multivesicular and residual bodies suggests that the ICPC might be involved in exchange, transport and oxidation processes, contributing to the excretory function at a subcellular level.     

Cytochemical studies on cytoplasmic RNA-associated basic proteins in oocytes,somatic cells,and ribosomes

Summary Cytochemical studies on oocytes from representatives of several animal groups, of somatic tissues known to possess high levels of cytoplasmic RNA, and of isolated ribosomes indicated that basic proteins associated with the ribosomes may be stained by the methods currently in use for demonstrating histones. These proteins were resistent to weak acid extraction in fixed material and were labile to acetylation, but pretreatment with hot 5% TCA caused a measurable increase in staining with a modified Sakaguchi reaction. Such proteins were not confined to oocytes, but could be demonstrated with the TCA-alkaline fast green method in mouse pancreas and liver cells, and by the ammoniacal-silver method in all cells with high levels of cytoplasmic RNA. Microspectrophotometric studies onAscidia nigra andClavelina picta oocytes indicated that the highest concentration of cytoplasmic basic proteins was found in smaller oocytes, but the concentration of alkaline fast green stainable cytoplasmic RNA-associated basic proteins decreased in a pattern that differed significantly from that of stainable levels of cytoplasmic RNA. The implications of these findings to current concepts in developmental biology were discussed.Nucleoplasmic basic proteins with high levels of arginine which are not associated with nucleic acids were encountered in oocytes of all the echinoderm species studied and in the mouse.This work was supported by a U. S. Public Health Service grant HD-1499-04 and a Career Development Award 5-K3-6176-04.Contribution number 382 of the Bermuda Biological Station.     

Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettre ascites tumor cells

Biochemical and autoradiographic evidence show both glycogen synthesis and the presence of glycogen synthase (UDP glucose [UDPG]: glycogen 4-alpha-D-glucosyltransferase; EC 2.4.1.11) in isolated nuclei of Ehrlich-Lettré mouse ascites tumor cells of the mutant subline HD33. 5 d after tumor transplantation, glycogen (average 5-7 pg/cell) is stored mainly in the cell nuclei. The activity of glycogen synthase in isolated nuclei is 14.5 mU/mg protein. At least half of the total cellular glycogen synthase activity is present in the nuclei. The nuclear glycogen synthase activity exists almost exclusively in its b form. The Km value for (a + b) glycogen synthase is 1 x 10(-3) M UDPG, the activation constant is 5 x 10(-3) M glucose-6-phosphate (Glc-6-P). Light and electron microscopic autoradiographs of isolated nuclei incubated with UDP-[1-3H]glucose show the highest activity of glycogen synthesis not only in the periphery of glycogen deposits but also in interchromatin regions unrelated to detectable glycogen particles. Together with earlier findings on nuclear glycogen synthesis in intact HD33 ascites tumor cells (Zimmermann, H.-P., V. Granzow, and C. Granzow. 1976. J. Ultrastruct. Res. 54:115-123), the results of tests on isolated nuclei suggest a predominantly appositional mode of nuclear glycogen deposition, without participation of the nuclear membrane system. In intact cells, synthesis of UDPG for nuclear glycogen synthesis depends on the activity of the exclusively cytoplasmic UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyltransferase; EC 2.7.7.9). However, we conclude that glycogen synthesis is not exclusively a cytoplasmic function and that the mammalian cell nucleus is capable of synthesizing glycogen.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Accumulation of cytoplasmic beta-catenin and nuclear glycogen synthase kinase 3beta in Epstein-Barr virus-infected cells

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. EBV establishes a latent infection and immortalizes and transforms B lymphocytes. Several latent proteins have profound effects on cellular growth, including activation of NF-kappaB, phosphatidylinositol 3'-OH kinase (PI3K) signaling, and notch signaling. Activation of PI3K can affect the activity of beta-catenin, the target of the wnt signaling pathway. Deregulation of beta-catenin is associated with a number of malignancies. To determine if beta-catenin is regulated by EBV infection, EBV-infected cells were examined for beta-catenin levels and localization. beta-Catenin was increased in EBV-positive tumor cell lines compared to EBV-negative lines, in EBV-infected Burkitt's lymphoma cell lines, and in EBV-transformed lymphoblastoid cell lines (LCL). In contrast to wnt signaling, EBV consistently induced the accumulation of beta-catenin in the cytoplasm but not the nucleus. The beta-catenin regulating kinase, glycogen synthase kinase 3beta (GSK3beta), was shown to be phosphorylated and inactivated in EBV-infected lymphocytes. Inactivated GSK3beta was localized to the nucleus of EBV-infected LCL. Neither the cytoplasmic accumulation of beta-catenin nor the nuclear inactivation of GSK3beta was affected by the inhibition of PI3K signaling. These data indicate that latent infection with EBV has unique effects on beta-catenin signaling that are distinct from activation of wnt and independent of its effects on PI3K.