Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes.

cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F. and Cohen, P.T.W. (1989) Biochim. Biophys. Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones. PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another. PPZ1 shows approx. 60% sequence identity to S. cerevisiae or mammalian PP1 and approx. 40% identity to S. cerevisiae or mammalian PP2A. These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.     

A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.     

The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.     

Molecular consequences of two formaldehyde-induced mutations in the alcohol dehydrogenase gene ofDrosophila melanogaster

Adhfn23 and Adhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene that codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) of Drosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis in Adhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast, Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As with Adhfn23, the deletion in Adhfn24 results in a change in the reading frame. Unlike Adhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on the Drosophila ADH mRNA levels and ADH protein levels are discussed.     

Protein serine/threonine phosphatases; an expanding family

Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57-69% identity) than PP1 (45-49% identity), while PPY and PPZ are more similar to PP1 (66-68% identity) than PP2A (44% identity). The cDNA for PP2Bw encodes a novel Ca2+/calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.     

The Yeast Translational Allosuppressor, Sal6: A New Member of the Pp1-like Phosphatase Family with a Long Serine-Rich N-Terminal Extension

The allosuppressor mutation, sal6-1, enhances the efficiency of all tested translational suppressors, including codon-specific tRNA suppressors as well as codon-nonspecific omnipotent suppressors. The SAL6 gene has now been cloned by complementation of the increased suppression efficiency and cold sensitivity caused by sal6-1 in the presence of the omnipotent suppressor sup45. Physical analysis maps SAL6 to chromosome XVI between TPK2 and spt14. The SAL6 gene encodes a very basic 549-amino acid protein whose C-terminal catalytic region of 265 residues is 63% identical to serine/threonine PP1 phosphatases, and 66% identical to yeast PPZ1 and PPZ2 phosphatases. The unusual 235 residue N-terminal extension found in SAL6, like those in the PPZ proteins, is serine-rich. The sal6-1 mutation is a frameshift at amino acid position 271 which destroys the presumed phosphatase catalytic domain of the protein. Disruptions of the entire SAL6 gene are viable, cause a slight growth defect on glycerol medium, and produce allosuppressor phenotypes in suppressor strain backgrounds. The role of the serine-rich N terminus is unclear, since sal6 phenotypes are fully complemented by a SAL6 allele that contains an in-frame deletion of most of this region. High copy number plasmids containing wild-type SAL6 cause antisuppressor phenotypes in suppressor strains. These results suggest that the accuracy of protein synthesis is affected by the levels of phosphorylation of the target(s) of SAL6.     

Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjected to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in lambda gt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxyl-terminal third is rich in alpha helices.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Yeast gene SRP1 (serine-rich protein). Intragenic repeat structure and identification of a family of SRP1-related DNA sequences

We have isolated and sequenced a yeast gene encoding a protein (Mr 24,875) very rich in serine (SRP) and alanine residues that accounted for 25% and 20% of the total amino acids, respectively. The SRP1 gene is highly expressed in culture conditions leading to glucose repression (Marguet & Lauquin, 1986), the amount of SRP1 mRNA representing about 1 to 2% of total poly(A)+ RNA. A repetitive structure of eight direct tandem repeats 36-base long, also reflected in the amino acid sequence, was found in the second half of the open reading frame. The consensus amino acid sequence of the repeat was Ser-Ser-Ser-Ala-Ala-Pro-Ser-Ser-Ser-Glu-Ala-Lys. Replacing the genomic copy of the cloned gene with a disrupted SRP1 gene indicated that the SRP1 gene was not essential for viability in yeast, but several SRP1-homologous sequences were found within the yeast genome, raising the possibility that the disrupted SRP1 gene is rescued by one of the other SRP-homologous sequences. Complete separation of yeast chromosomes by contour-clamped homogeneous field electrophoresis indicated that, apart from chromosome V, which carries the SRP1 gene, 12 chromosomes have SRP-related sequences with various degrees of homology. These sequences were located on chromosomes XV, VII and XI under stringent conditions of hybridization (tm -20 degrees C), and observed on chromosomes I, II, III, IV, VI, VIII, X, XI and XII, only under low-stringency conditions (tm -40 degrees C). Northern blot analysis of both the wild type and SRP1-disrupted strains indicated that along with SRP1 at least one more member of the SRP family was transcribed to a 0.7 kb (1 kb = 10(3) bases) polyadenylated RNA species clearly distinct from the SRP1-specific mRNA (1 kb long). Analyses of the SRP1 repeat domain suggested a model for the divergent evolution of the repeats in the SRP1 sequence.     

Identification of the tetracycline resistance promoter and repressor in transposon Tn10.

The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.     

Type 2C protein phosphatases in fungi

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.     

A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (B?hni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.     

A barbiturate-regulated protein binding to a common sequence in the cytochrome P450 genes of rodents and bacteria

Analyses of the 5' regulatory sequences of genes encoding barbiturate-inducible cytochromes P450BM-1 and P450BM-3 from Bacillus megaterium and of the 5' sequences of genes for barbiturate-inducible P450b and P450e of the rat revealed a string of 17 base pairs in each of the genes that shared a high degree of sequence identity. Labeled oligonucleotide probes of each of these four sequences were tested in gel retardation assays with protein obtained from B. megaterium grown either in the presence or absence of barbiturates or with protein from nuclear extracts from livers of rats left untreated or injected with phenobarbital. Each of the four 17-mers bound strongly to a single protein from bacteria grown in the absence of barbiturates, but this binding was dramatically reduced with protein from pentobarbital- or phenobarbital-grown cells. Conversely, the probes complexed weakly to one protein band from nuclear extracts from untreated rats but much more strongly with protein from phenobarbital-treated rats. Similar effects could be obtained by prolonged incubation with phenobarbital of either soluble protein from the bacteria grown in the absence of barbiturates or nuclear extract protein from untreated rats. Deletion analysis of the 5'-flanking region of the P450BM-1 gene of B. megaterium revealed a putative repressor binding site located within a 24-base pair DNA segment that included the 17-base pair sequence involved in barbiturate-regulated protein binding.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Isolation and sequence of a rat chymotrypsin B gene

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.