Light and the recovery from heat shock induce the synthesis of 38 kDa mitochondrial proteins in Neurospora crassa

The effect of light on the protein synthesis pattern in the mitochondria of Neurospora crassa was examined by in vivo labelling with [³⁵S]-methionine and two-dimensional gel electrophoresis. A brief 5-min illumination induced the rapid and transient synthesis of a 38-kDa protein. White collar-mutants were not stimulated to synthesize this protein by light. A protein of a similar molecular weight and isoelectrical point was synthesized during recovery from heat shock.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Efficient protein folding and quality control are essential for unperturbed cell viability. Defects in these processes may lead to production of aberrant polypeptides that are either degraded leading to “loss-of-function” phenotypes, or deposited in or outside cells leading to “gain-of-toxic-function” phenotypes. Elucidation of molecular mechanisms regulating folding and quality control of newly synthesized polypeptides is therefore of greatest interest. Here we describe protocols for metabolic labelling of transfected/infected mammalian cells with [³⁵S]-methionine and [³⁵S]-cysteine, for immunoisolation from detergent extracts of the selected model proteins and for the investigation of the model polypeptide’s intracellular fate in response to chaperone-deletions or to cell exposure to folding or degradation inhibitors.     

Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe

Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins. We have modified this technique for the isolation of plant calmodulin-binding proteins. [³⁵S]-methionine was used instead of the inorganic [³⁵S]-sulfate, or¹²⁵I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large number of filters. Here we describe in detail a procedure for the production and purification of [³⁵S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The [³⁵S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II that was previously isolated using a [¹²⁵I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive clone encoding a calcium-dependent calmodulin-binding protein was isolated.     

Induction of novel protein synthesis by opsonizedHistoplasma capsulatum ingested by murine peritoneal macrophages

It is known thatHistoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closly related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form ofH. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingestedH. capsulatum with [³⁵S]-methionine labeling. Cycloheximide at 5 to 10 µg/ml was used to preferentially inhibit macrophage uptake of [³⁵S]-methionine without affectingH. capsulatum uptake. Protein synthesis byH. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killedH. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingestedH. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingestedH. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

MONOCLONAL ANTIBODY RAISED AGAINST HUMAN MITOTIC CYCLIN B1IDENTIFIES CYCLIN B-LIKE MITOTIC PROTEINS IN SYNCHRONIZED ONION (ALLIUM CEPA L.) ROOT MERISTEM

Cyclin B-like mitotic proteins have been detected in synchronized Allium cepa L. root tip cells by using mouse monoclonal anti-cyclin B₁antibody raised against human cyclin B₁Immunoblot shows two closely placed isoforms of cyclin B-like proteins having an apparent molecular weight around 54 kDa. In vivo [³⁵S]-methionine labelling followed by immunoprecipitation and autoradiography indicates that cyclin B-like proteins are mainly synthesized in the G2 phase of the cell cycle and destroyed in late mitosis. Immunoblotting data depict that the level of cyclin B-like proteins reaches the maximum at the late G2 to early M phase; and it becomes degraded in the late hours of mitosis. Moreover, the cyclin B isoforms are stabilized in colchicine-arrested metaphase cells as already reported in animal cells.     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.     

Identification of Ceramide Binding Proteins in Neuronal Cells: A Critical Point of View

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[³⁵S]-2-thioacetyl-d-erythro-C₁₈-sphingosine ([³⁵S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

In maize,glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes

Summary Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [³⁵S]-methionine or [³⁵S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.     

Heterogeneity of sulphur content in the storage proteins of pea cotyledons

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [³⁵S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.     

Characterization of protein synthesis by isolated rice mitochondria

Summary Bacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [³⁵S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.     

Methionine-rich hexamerin and arylphorin as precursor reservoirs for reproduction and metamorphosis in female luna moths

The storage proteins of Lepidoptera include a pair of methionine-rich hexamerins (MtH) that are more abundant in female pupae than in males. Their inferred support of female reproduction could be achieved either by enhancing general pools of amino acids, or by hydrolyzing MtH at times and/or sites that direct its constituents to the synthesis of egg proteins. The two models were tested in Actias luna, a saturniid moth that makes its eggs during adult development. MtH and arylphorin (ArH), the third major storage protein of this species, were labeled metabolically with [³⁵S]-methionine and [³H]-leucine, and injected individually into wandering stage caterpillars. Isotope distributions at eclosion indicated that both hexamerins supported egg formation as well as adult tissue protein synthesis. In the absence of evidence for targeting, MtH appears to support egg formation in A. luna by enhancing the amino acid pools derived from ArH. Analysis of ³⁵S labeling and of ³⁵S/³H ratios indicated, however, that ArH is consumed over a period that extends somewhat later in adult development than MtH. Differences in timing should prove to be much greater in Lepidoptera that delay egg formation until after eclosion. © 1996 Wiley-Liss, Inc.     

Plant nuclear protein p 56 and its elicitor-dependent transient biosynthesis

Summary Pulse labelling experiments with [³⁵S]L-methionine were performed to determine the rate of protein synthesis. Treatment of cultured cells of peanut with fungal cell wall led to a drastic increase in the de novo synthesis of particular proteins in the cytosol, the endoplasmic reticulum and the extracellular compartment. In the nucleus, a single newly synthesized protein, designated p 56, was detectable upon elicitation by fungal elicitor. Pulse labelling with [³⁵S]L-methionine for 1h was applied at various times following elicitation. The time course of p 56 biosynthesis was transient and the maximum of p 56 de novo synthesis preceded the one of the cytosolic protein stilbene synthase. The preferential de novo synthesis and transfer of p 56 to the nucleus, only briefly before the elicitortriggered signal chain causes the activation of nuclear defence genes, makes it a good candidate as member of the signal transduction machinery to the nucleus. p 56 was further characterized by its size as N-octyl--D-glucoside micelle. Selective solubilization experiments showed that p 56 is a hydrophobic, not salt extractable protein rather well protected against partial proteolysis.Abbreviation CHAPS 3-(cholamidopropyl-dimethylammonio)-propane-1-sulfonate     

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

Extraction by lithium chloride of hydroxyproline-rich glycoproteins from intact cells of Chlamydomonas reinhardii

A procedure has been developed to isolate and analyse the cell-wall glycoproteins of Chlamydomonas reinhardii. Under appropriate conditions, cell-wall glycoproteins can be quantitatively extracted from intact cells by aqueous LiCl. Although proteins and glycoproteins, which are presumably not related to the cell wall, are coextracted with the cell-wall subunits, these components can be readily identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as demonstrated by comparative analysis of LiCl-extracts from wild-type cells and the cell-wall-deficient mutant CW-15. Apart from the high-molecular-weight cell-wall components, two glycoproteins with apparent molecular weights (M_rs) of 36000 and 66000 were found to be present in LiCl-extracts of wild-type cells but absent in LiCl-extracts from the cell-wall-less mutant. Pulse-labeling experiments with [³H]proline and [³⁵S]methionine revealed that the LiCl-extracts contained — in addition to the well-known cell-wall subunits — proteins of lower molecular weight, which are also preferentially labeled with [³H]proline. Protein components with M_rs of 68000, 44000, 36000, 26000 and 22000 were found to be more strongly labeled with [³H]proline than with [³⁵S]methionine, whereas protein components with M_rs of 57000 and 52000 were more prominent after labeling with [³⁵S]methionine. The portion of cell-wall subunits within the total amount of proteins extracted by LiCl was calculated to be at least 10% on the basis of the amount of hydroxyproline. Self-assembly of cell walls could be demonstrated after dialysis against water of a mixture of crude LiCl-extract and purified, insoluble, inner wall layers. Cell-wall glycoproteins could be enriched by gel exclusion chromatography of crude LiCl-extracts on Sepharose CL-4B columns equilibrated with 1 mol l^-1 LiCl.Abbreviations EDTA ethylenediaminetetraacetic-acid - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff's reagent - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Proteins of the Drosophila melanogaster male reproductive system: Two-dimensional gel patterns of proteins synthesized in the XO,XY, and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins

Testes and paragonial glands of Drosophila melanogaster wild-type males were labeled in vitro using [³⁵S]methionine, and the proteins synthesized were analyzed by 2-dimensional gel electrophoresis. Testes and paragonial glands were also labeled in vivo by feeding male larvae ³⁵S-labeled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. [³H]Proline was also used to label testes, and no significant differences from the ³⁵S pattern were noted. Testes and paragonial glands from XO and XYY males were labeled in vitro with [³⁵S]methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Pure sperm were dissected manually from in vivo labeled males and the proteins analyzed. Ninety-two proteins were detected, which were all synthesized in comparable amounts by XO, XY, and XYY males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational or regulatory proteins present only in very small amounts in the adult testes. ³⁵S-labeled males were also mated to unlabeled females and the transferred proteins analyzed on two-dimensional PAGE. The contributions of the testis and paragonial gland to the ejaculate were determined.This work was supported by a grant from the Medical Research Council of Canada and by the U.S. National Institutes of Health (Grant No. GM-22753). J. I. B. is the recipient of a Medical Research Council of Canada studentship.     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum