Isolation and characterization of an allelic cDNA for human muscle fructose-1,6-bisphosphatase

By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose-1,6-bisphosphatase was found to be active in cell-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.     

Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.     

Changes of an androgen-dependent nuclear protein during functional differentiation and by dedifferentiation of the dorsolateral prostate of rats

In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S. cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide. Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S. cerevisiae. The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.     

Distribution of a COOH-terminal amino acid extension of liver fructose-1,6-bisphosphatase among rodent species

We have recently established from sequence analysis that rat liver fructose-1,6-bisphosphatase contains a 24-26 residue extension beyond the COOH-terminal amino acid of other mammalian fructose-1,6-bisphosphatases that results in an increased subunit molecular weight (Rittenhouse et al. (1983) J. Biol. Chem. 258, 7648-7652). In the present work the distribution of the COOH-terminal extension of fructose-1,6-bisphosphatases was tested by subunit molecular weight analysis of the enzyme immunoprecipitated from liver extracts. Of all rodent species tested, including several Muridae other than Rattus; only the enzyme from animals of the genus Rattus was found to have the extension. Further studies on the distribution of the enzyme extension could provide a simple tool to study the phylogeny of the genus Rattus.     

Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

The primary structure of rat ribosomal protein S6

The amino acid sequence of rat ribosomal protein S6, the major phosphoprotein in the organelle, was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the sequence of amino acids in portions of the protein. Ribosomal protein S6 contains 249 amino acids and has a molecular weight of 28,683. The protein has 15 seryl residues; 7 are located in the carboxyl-terminal sequence of 15 amino acids and probably include most if not all of the residues that are phosphorylated. There are related repeated sequences of 10 amino acids each that occur at four separate positions in S6 and that are very basic. Rat S6 is homologous to Saccharomyces cerevisiae S10 (the extent of the identity is 75%) and most likely also to Schizosaccharomyces pombe S6.     

Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein

Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.     

Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe

The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

Abstract The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp , encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO₂. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp .     

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.