Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.

Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.     

Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase.

Phosphotyrosine-Sepharose 4B was synthesized and used to purify L-cell alkaline phosphatase. Antibodies to this enzyme interacted with the alkaline phosphatase of strains A-1-2 and A-3-3, mutants that express the enzyme constitutively. This and thermal stability studies suggest that these mutants contain the same alkaline phosphatase isozyme as their parent strain.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis.Their phosphatase activity and the corresponding solution conformation were examined.Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immunosuppressive drugs with CN.The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN.Furthermore,circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein.Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN,and had no effects on the phosphatase activity of mutants in Loop7 region,which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN.Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs.The component of Chinese drug,ZIP1,could directly inhibit both CN and CN mutants without drug binding protein.These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA.It is valuable to further study the inhibition by ZIP1.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi.

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.     

Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli

We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm. Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations. Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus. Included in the latter class are 47 mutants which are characterized in detail in this report. To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector. DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export. Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.     

Inorganic phosphate transport in Escherichia coli: involvement of two genes which play a role in alkaline phosphatase regulation

Two classes of alkaline phosphatase constitutive mutations which comprise the original phoS locus (genes phoS and phoT) on the Escherichia coli genome have been implicated in the regulation of alkaline phosphatase synthesis. When these mutations were introduced into a strain dependent on a single system, the pst system, for inorganic phosphate (P(i)) transport, profound changes in P(i) transport were observed. The phoT mutations led to a complete P(i) (-) phenotype in this background, and no activity of the pst system could be detected. The introduction of the phoS mutations changed the specificity of the pst system so that arsenate became growth inhibitory. Changes in the phosphate source led to changes in the levels of constitutive alkaline phosphatase synthesis found in phoS and phoT mutants. When glucose-6-phosphate or l-alpha-glycerophosphate was supplied as the sole source of phosphate, phoT mutants showed a 3- to 15- fold reduction in constitutive alkaline phosphatase synthesis when compared to the maximal levels found in limiting P(i) media. However, these levels were still 100 times greater than the basal level of alkaline phosphatase synthesized in wild-type strains under these conditions. The phoS mutants showed only a two- to threefold reduction when grown with organic phosphate sources. The properties of the phoT mutants selected on the basis of constitutive alkaline phosphatase synthesis were similar in many respects to those of pst mutants selected for resistance to growth inhibition caused by arsenate. It is suggested that the phoS and phoT genes are primarily involved in P(i) transport and, as a result of this function, play a role in the regulation of alkaline phosphatase synthesis.     

Metabolic and genetic control of isoenzyme spectrum of alkaline phosphatase inEscherichia coli

Three proteins possessing alkaline phosphatase activity were detected in a fraction of periplasmic material ofEscherichia coli K-10 and its mutants with constitutive synthesis of alkaline phosphatase. They also showed acid phosphatase, pyrophosphatase and ATPase activities. Through the use of phosphatase-negative mutants it was shown that these proteins were the products of a single structural gene and therefore represented alkaline phosphatase isozymes. The numbers of enzyme isoforms and possibly the spectrum of their phosphohydrolase activities were controlled by exogenous orthophosphate and depended on the integrity of regulator genes for alkaline phosphatase.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA : Identification of the Structural Gene for Repressible Alkaline Phosphatase

Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (GLEASON and METZENBERG 1974). The five new mutants, as well as three previously isolated by GLEASON and METZENBERG (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.     

Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi

Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.Chercheur qualifié du Fonds National Belge de la Recherche Scientifique.     

The responses of wild-type and ABA mutant Arabidopsis thaliana plants to phosphorus starvation

The growth patterns of plants subjected to phosphorus starvation resemble those caused by treatment with ABA, suggesting that ABA could mediate the response of the plant to phosphorus starvation. We examined the role of ABA in phosphorus stress by comparing growth and biochemical responses of Arabidopsis thaliana ABA mutants aba-1 and abi2-1 to those of wild-type plants. We first characterized acid phosphatase production of wild-type Arabidopsis in response to phosphorus starvation. We found that several acid phosphatase isozymes are present in roots and shoots, but only a subset of these isozymes are induced by phosphorus stress, and they are induced in both organs. Production of acid phosphatase in response to phosphorus stress was not affected by the aba-1 or abi2-1 mutations. Low phosphorus also resulted in decreased growth of both wild-type and ABA mutant plants, and the root-to-shoot ratio was increased in both wild type and mutants. Anthocyanins accumulated in response to phosphorus stress in both wild-type and mutant plants, but the increase was reduced in the aba-1 mutant. Thus, two different ABA mutants responded normally in most respects to phosphorus stress. Our data do not support a major role for ABA in coordinating the phosphorus-stress response.     

Regulation of the neutral phosphatase in Chlamydomonas reinhardi: An immunogenetic study of wild-type and mutant strains

In Chlamydomonas reinhardi, the activity of the neutral phosphatase considerably increases when the cells are grown in the absence of inorganic phosphate (Pi). A comparative immunological study of cells grown on media containing Pi or not indicated that the neutral phosphatase was synthesized de novo. Ten mutants lacking the neutral phosphatase and distributed among three genetic loci (PD2, PD3, PD24) were investigated for their ability to produce cross-reacting material (CRM) antigenically related to the wild enzyme. All mutants were shown to form much less CRM than the wild-type strain. It is proposed that the three genes are involved in the regulation of neutral phosphatase synthesis.     

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.     

Isolation and Study of Mutants Lacking a Derepressible Phosphatase in CHLAMYDOMONAS REINHARDI

In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa)) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of elctropherograms form WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas: in addition tothe neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.     

Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants

We have isolated 2 new pleiotropic mutants of Pseudomonas aeruginosa strain PAO with defective secretion of extracellular proteins (Xcp mutants). One of these mutants was compared to 2 different, previously isolated secretion mutants. All had similar phenotypes and were unable to release at least 4 exoproteins (lipase, elastase, alkaline phosphatase, and phospholipase C), whilst alkaline protease was still secreted. The exoproteins appeared to be blocked in the periplasmic space. No difference in molecular weight was detected between cell-bound forms of elastase and alkaline phosphatase in the different mutants and the corresponding extracellular forms from the wild-type strain. Genetic mapping showed that the mutations were located in the 55′ region of the chromosome.     

Improved properties of baker's yeast mutants resistant to 2-deoxy-D-glucose

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-D-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-D-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.