Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin

The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000–20,000.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. Received: 21 January 1997 / Accepted: 21 June 1997     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

水稻rab5B基因在大肠杆菌中的表达、纯化和GTP结合分析

Rab5B类蛋白因为其编码产物的N端具有特殊结构而被认为是一类特殊的蛋白质.水稻rab5B基因Osrab5B是这类蛋白质基因在单子叶植物中的首例发现.将Osrab5B基因的编码序列按正确读码框重组到具有谷胱甘肽硫转移酶（glutathione S-transferase, GST）融合标签的pGEX-4T1表达载体中,转化大肠杆菌,获得了稳定表达目标融合蛋白的菌株,经GSTrap^TM柱纯化,获得了纯化的目标融合蛋白.GTP结合试验表明,在原核细胞中表达出的GST-OsRab5B融合蛋白具有体外结合GTP的能力.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Bidirectional insulin granule turnover in the submembrane space during K(+) depolarization-induced secretion

Like primary mouse islets, MIN6 pseudoislets responded to the depolarization by 40 mm KCl and the resulting increase in the free cytosolic Ca(2+) concentration ([Ca(2+) ](i) ) with a massive increase in insulin secretion, whereas 15 mm KCl had little effect in spite of a clear increase in [Ca(2+) ](i) . Analysis of insulin-enhanced green fluorescent protein (EGFP)-labeled granules in MIN6 cells by total internal reflection fluorescence (TIRF) microscopy showed that 40 mm KCl increased the number of short-term resident granules (<1 second presence in the submembrane space), while the total granule number and the number of long-term resident granules decreased. The rates of granule arrival at and departure from the submembrane space changed in parallel and were two orders of magnitude higher than the release rates, suggesting a back-and-forth movement of the granules as the primary determinant of the submembrane granule number. The effect of 15 mm KCl resembled that of 40 mm but did not achieve significance. Both 15 and 40 mm KCl evoked a [Ca(2+) ](i) increase, which was antagonized by 10 μm nifedipine. Nifedipine also antagonized the effect on secretion and on granule number and mobility. In conclusion, during KCl depolarization L-type Ca(2+) channels seem to regulate two processes, insulin granule turnover in the submembrane space and granule exocytosis.     

Desensitization of the insulin-secreting beta cell.

In human diabetes, inherent impaired insulin secretion can be exacerbated by desensitization of the beta cell by chronic hyperglycemia. Interest in this phenomenon has generated extensive studies in genetic or experimentally induced diabetes in animals and in fully in vitro systems, with often conflicting results. In general, although chronic glucose causes decreased beta-cell response to this carbohydrate, basal response and response to alternate stimulating agents are enhanced. Glucose-stimulated insulin synthesis can be increased or decreased depending on the system studied. Using a two-compartment beta-cell model of phasic insulin secretion, a unifying hypothesis is described which can explain some of the apparent conflicting data. This hypothesis suggests that glucose-desensitization is caused by an impairment in stimulation of a hypothetical potentiator singularly responsible for: 1) some of the characteristic phases of insulin secretion; 2) basal release; 3) potentiation of non-glucose stimulators; and 4) apparent "recovery" from desensitization. Review of some of the pathways that regulate insulin secretion suggest that phosphoinositol metabolism and protein kinase-C production are regulated similarly to the theoretical potentiator and their impairment is a major contributor to glucose desensitization in the beta cell.     

Apoplastic exosome-like vesicles: A new way of protein secretion in plants?

The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.     

Peptide-chain elongation in eukaryotes

The elongation phase of translation leads to the decoding of the mRNA and the synthesis of the corresponding polypeptide chain. In most eukaryotes, two distinct protein elongation factors (eEF-1 and eEF-2) are required for elongation. Each is active as a complex with GTP. eEF-1 is a multimer and mediates the binding of the cognate aminoacyl-tRNA to the ribosome, while eEF-2, a monomer, catalyses the movement of the ribosome relative to the mRNA. Recent work showing that bacterial ribosomes possess three sites for tRNA binding and that during elongation tRNAs may occupy hybrid sites is incorporated into a model of eukaryotic elongation. In fungi, elongation also requires a third factor, eEF-3. A number of mechanisms exist to promote the accuracy or fidelity of elongation: eEF-3 may play a role here. cDNAs for this and the other elongation factors have been cloned and sequenced, and the structural and functional properties of the elongation factors are discussed. eEF-1 and eEF-2 can be regulated by phosphorylation, and this may serve to control rates of elongationin vivo.Abbreviations eEF eukaryotic elongation factor- - PKC protein kinase C     

Chloroplast protein import inhibition by a soluble factor from wheat germ lysate

Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.     

Remodelling of VipA/VipB tubules by ClpV‐mediated threading is crucial for type VI protein secretion

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N‐terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel‐like structures that are converted by a threading activity of ClpV into small complexes. ClpV‐mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion.     

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine

Summary Membrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively.Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, -subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [-³²P]ATP.By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any.The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCI) concentrations up to physiological levels (140 mM KCI, 20 mM NaCI); i.e. CS-and MS-catalyzed phosphorylations are strongly inhibited by 75–150 mM KCI (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected.In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected.Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.     

Peptidase N encoded by Salmonella enterica serovar Typhimurium modulates systemic infection in mice

The cytosolic protein degradation pathway, involving ATP-dependent proteases and ATP-independent peptidases, is important for modulating several cellular responses. The involvement of pathogen-encoded ATP-dependent proteases is well established during infection. However, the roles of ATP-independent peptidases in this process are not well studied. The functional role of Peptidase N (PepN), an ATP-independent enzyme belonging to the M1 family, during systemic infection of mice by Salmonella enterica serovar Typhimurium (Salmonella typhimurium) was investigated. In a systemic model of infection, the number of CFU of S. typhimurium containing a targeted deletion in peptidase N (DeltapepN), compared with wild type, was significantly higher in the lymph node and spleen. In addition, S. typhimurium replicated in the thymus and greatly reduced the number of immature CD4(+)CD8(+) thymocytes in a dose- and time-dependent manner. Strains lacking or overexpressing pepN were used to show that the reduction in the number of thymocytes, but not lymph node cells, depends on a critical number of CFU. These findings establish a role for PepN in reducing the in vivo CFU of S. typhimurium during systemic infection. The implications of these results, in the context of the roles of proteases and peptidases, during host-pathogen interactions are discussed.     

Molecular Cloning of cDNA for BRab from the Brain of Bombyx mori and Biochemical Properties of BRab Expressed in Escherichia coli

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [³⁵S]-GTPγS and [³H]-GDP with association constants of 1.5×10⁶ M^-1 and 0.58×10⁶ M^-1, respectively. The binding of [³⁵S]-GTPγS was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl₂, bound [³⁵S]-GTPγS and [³H]-GDP were exchanged with GTPγS most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.