Synthesis of Escherichia coli outer membrane OmpA protein in yeasts

Saccharomyces cerevisiae was transformed with the Escherichia coli ompA gene coding for an outer membrane protein. Yeast transformants containing the pYTLJ101 plasmid, consisting of the ompA gene cloned in pSC101 and the HindIII-3 fragment of 2-μm DNA, express the foreign membrane protein. The protein synthesized in yeast has an M_r value very similar if not identical to that of the mature E. coli protein. The expressed protein is present in yeast mitochondrial and plasma membrane fractions. The yeast cell can tolerate about 250 molecules of the foreign membrane protein per cell, although the transformants show altered growth kinetics.     

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M_r), KP-2 (pI 4.7/20,700 M_r) and KP-10 (pI 4.7/25,000 M_r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca²⁺-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca²⁺-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.     

Cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of Bacillus subtilis phage φ 29

The φ 29 DNA restriction fragment HindIII-D, shown to contain gene 10 coding for the connector protein, has been cloned in plasmid pPLc28 under the control of the p_L promoter of phage λ. After heat induction to inactivate the λ repressor, a protein with the electrophoretic mobility of the connector protein p 10 was synthesized, accounting for about 30 % of the total Escherichia coli protein after 3 h of induction. The 2205 nucleotide-long sequence of the cloned HindIII-D fragment has been determined. The sequenced region has an ORF coding for a protein of M_r 35881 that was shown to correspond to the connector protein by determination of the ammo-terminal sequence of purified protein p10. Features of the nucleotide sequence and the amino acid sequence of protein p10 are discussed.     

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a λgt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M_r of 30 227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosomespecific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95–102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29–38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.     

Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells

Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072–2076]. Mouse cells normally sensitive to 100 μg/ml Hm were transformed with these plasmids and selected in 200 μg/ml Hm. Transformants resistant to as much as l mg/ml Hm and 500 μg/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm amino-cyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high M_r DNA isolated from transformed cells.     

Stress relaxation function of bone and bone collagen

Relaxation Young's and shear moduli of bovine bone and bone collagen were investigated. It was found that each relaxation process observed had two stages, which were referred to as process I and process II in order of time. Process II was described by a simple exponential decay while process I was not. The Kohlrausch-Williams-Watts (KWW) function, ψ(t) = exp[t/τ₁)^B] (0 < B < 1), was found to be suitable to describe process I. The normalized relaxation modulus, M_r(t), was expressed by the combination of the simple exponential type relaxation function and the KWW function

M_r=A₁exp[−(t/τ₁)^B]+A₂exp[(t/τ₁)](0<B1)

On the basis of this equation, the relaxation mechanism in bone and bone collagen was identified. According to the model proposed for the KWW relaxation function, the stress relaxation process in bone was considered to be governed by viscoelastic properties of matrix collagen fiber. The model for the KWW relaxation function requires the disordered glassy structure of collagen fiber, which is consistent with the results of the structural investigations.     

The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase

The F_O portion of the mitochondrial ATP synthase contains a range of different subunits in bacteria, yeast and mammals. A search of the Arabidopsis genome identified sequence orthologs for only some of these subunits. Blue native polyacrylamide gel electrophoresis separation of Arabidopsis mitochondrial respiratory chain complexes revealed intact F₁F_O, and separated F₁ and F_O components. The subunits of each complex were analysed by mass spectrometry and matched to Arabidopsis genes. In the F₁F_O complex a series of nine known subunits were identified along with two additional proteins matching the predicted products of the mitochondrial encoded orfB and orf25 genes. The F₁ complex contained the five well-characterised F₁ subunits, while four subunits in the F_O complex were identified: subunit 9, d subunit, and the orfB and orf25 products. Previously, orfB has been suggested as the plant equivalent of subunit 8 based on structural and sequence similarity. We propose that orf25 is the plant b subunit based on structural similarity and its presence in the F_O complex. Chimerics of orf25, orfB, subunit 9 and subunit 6 have been associated with cytoplasmic male sterility in a variety of plant species, our additional findings now place all these proteins in the same protein complex.     

A 56,000 Mr phosphoseryl protein in PC12 cell lysates strongly associates with protein-A sepharose beads and was observed in immune complex kinase assays for PP60c-src

Using an immune complex kinase assay to measure pp60c-src kinase activity, we have identified a 56,000 M_r protein (p56) from PC12 cell lysates that co-purified with pp60c-src by strong association with protein-A sepharose beads. The p56 protein was strongly phosphorylated on serine but no tyrosine or threonine phosphorylation was evident. However, pp60c-src was strongly phosphorylated on tyrosine, weakly phosphorylated on serine with no observed threonine phosphorylation. P56 was not a proteolytic breakdown product of pp60c-src, since it was neither tyrosine phosphorylated nor was it recognized by anti-src antibody. P56 was also not recognised by other antibodies to 56kD signalling molecules such as p56lck. The identity of p56 awaits further investigation but its appearance in immunoprecipitates of pp60c-src using protein-A sepharose beads is of interest but complicates the interpretation of results from immune complex kinase assays in PC12 cells.     

Cell wall polysaccharide interactions in maize bran

Sequential extractions with alkali have been carried out in order to study the nature of linkages which hold heteroxylans in maize bran cell walls. Treatment with 0.5 sodium hydroxide at 30 °C for 2 h released all the phenolic acids (p-coumaric, ferulic, and diferulic) but extracted only ˜30% of heteroxylans (S₁); further treatment with 1.5 potassium hydroxide at 100 °C for 2 h released the remaining heteroxylans (S₂). The heteroxylans from S₁ and S₂ had a similar neutral sugar composition and structure, but their weight average molecular weights were 270 kDa (M_w/M_n = 2) and 370 kDa (M_w/M_n = 2.8), respectively. Proteins (5%) are found with polysaccharides from S₁ and S₂, with different amino acid composition. The results suggest that covalent linkages through phenolic acids are only partly responsible for the associations of maize bran heteroxylans in the cell wall and that linkages to structural cell wall proteins were probably the main cause of heteroxylan insolubility.     

Protein complexity, gene duplicability and gene dispensability in the yeast genome

Using functional genomic and protein structural data we studied the effects of protein complexity (here defined as the number of subunit types in a protein) on gene dispensability and gene duplicability. We found that in terms of gene duplicability the major distinction in protein complexity is between hetero-complexes, each of which includes at least two different types of subunits (polypeptides), and homo-complexes, which include monomers and complexes that consist of only subunits of one polypeptide type. However, gene dispensability decreases only gradually as the number of subunit types in a protein complex increases. These observations suggest that the dosage balance hypothesis can explain well gene duplicability of complex proteins, but cannot completely explain the difference in dispensabilities between hetero-complex subunits. It is likely that knocking out a gene coding for a hetero-complex subunit would disrupt the function of the whole complex, so that the deletion effect on fitness would increase with protein complexity. We also found that multi-domain polypeptide genes are less dispensable but more duplicable than single-domain polypeptide genes. Duplicate genes derived from the whole genome duplication event in yeast are more dispensable (except for ribosomal protein genes) than other duplicate genes. Further, we found that subunits of the same protein complex tend to have similar expression levels and similar effects of gene deletion on fitness. Finally, we estimated that in yeast the contribution of duplicate genes to genetic robustness against null mutation is approximately 9%, smaller than previously estimated. In yeast, protein complexity may serve as a better indicator of gene dispensability than do duplicate genes.     

Effects of anoxia and the mitochondrion on expression of aerobic nuclear COX genes in yeast: evidence for a signaling pathway from the mitochondrial genome to the nucleus

Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.     

Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes

Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

互作蛋白质与复合物亚基的基因表达相关性比较分析

利用在多种应激条件下酵母的基因表达谱数据 ,分别计算互作蛋白质及复合物亚基编码基因的表达相关性。结果发现 ,相对于随机对照组 ,互作蛋白质的编码基因与蛋白质复合物的编码基因表达相关性均显著 (P <0 .0 1) ,即互作蛋白质及复合物亚基有共表达的倾向。通过比较 ,进一步发现蛋白质复合物亚基的基因表达相关性显著高于互作蛋白质的基因表达相关性 (P <0 .0 1) ,这与复合物亚基之间功能联系强于定义不甚确切的互作蛋白之间功能联系现象吻合。     

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.