DPM2 regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate

Biosynthesis of glycosylphosphatidylinositol and N-glycan precursor is dependent upon a mannosyl donor, dolichol phosphate-mannose (DPM). The Thy-1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis. The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis. Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells. DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1. Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase. Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2. Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2.     

Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe

Dolichol phosphate mannose (Dol-P-Man), formed upon transfer of Man from GDPMan to Dol-P, is a mannosyl donor in pathways leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of protein. Dol-P-Man synthase is an essential protein in Saccharomyces cerevisiae. We have cloned cDNAs encoding human and Schizosaccharomyces pombe proteins that resemble S. cerevisiae Dol-P-Man synthase. Disruption of the gene for the S. pombe Dol-P-Man synthase homolog, dpm1(+), is lethal. The known Dol-P-Man synthase sequences can be divided into two classes. One contains the S. cerevisiae, Ustilago maydis, and Trypanosoma brucei enzymes, which have a COOH-terminal hydrophobic domain, and the other contains the human, S. pombe, and Caenorhabditis synthases, which lack a hydrophobic COOH-terminal domain. The two classes of synthase are functionally equivalent, because S. cerevisiae DPM1 and its human counterpart both complement the lethal null mutation in S. pombe dpm1(+). The findings that Dol-P-Man synthase is essential in yeast and that the Ustilago and Trypanosoma synthases are in a different class from the human enzyme raise the possibility that Dol-P-Man synthase could be exploited as a target for inhibitors of pathogenic eukaryotic microbes.     

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.     

Molecular identification of a putative human hyaluronan synthase

To identify the putative mammalian hyaluronan synthase, we cloned a human cDNA that is related to the Streptococcus hyaluronan synthase (HasA) and the Xenopus developmental protein DG42 which has been shown to have chitin synthase activity. The cDNA, for which we propose the name Has2, encodes a novel protein with a predicted molecular mass of 63.6 kDa. Has2 shows 55% amino acid identity with Xenopus DG42 and 52% identity with the mouse HAS protein, another putative hyaluronan synthase recently reported by Itano and Kimata (Itano, N., and Kimata, K. (1996) J. Biol. Chem. 271, 9875-9878). The deduced primary structure revealed the presence of several hydrophobic stretches which can form multiple transmembrane domains. It also demonstrated the complete conservation of amino acid residues that are known to be critical for N-acetylglucosaminyltransferase activity of yeast chitin synthase. When the Has2 cDNA was transfected into human 293 and Chinese hamster ovary cells, the production of hyaluronan in the transfected cells increased up to 34- and 9-fold, respectively. Strong expression of Has2 mRNA was observed in exponentially proliferating human IMR-90 fibroblasts but not in growth-arrested IMR-90 cells. These results suggest that the Has2 protein is a crucial component of the human hyaluronan synthase system.     

Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.     

Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.     

Alpha 1,6-linked fucose affects the expression and stability of polysialic acid-carrying glycoproteins in Chinese hamster ovary cells

To determine the effect of alpha1,6-linked fucose modification of N-glycans on the expression of polysialic acids (PSAs), the expression of PSAs in a fucose-lacking mutant of Chinese hamster ovary (CHO) cells, Lec13, was compared with that in CHO K1 cells. PSA synthase activity in these cells and the antennary structures of N-glycans associated with the neural adhesion molecule (NCAM), which is a major PSA-carrying glycoprotein, did not differ between the two types of cells. Metabolic labeling of cells with [3H]glucosamine for 48 h followed by immunoprecipitation with anti-PSA monoclonal antibodies revealed that the amount of labeled PSA-carrying glycoproteins obtained from Lec13 cells was 10-times less than that from K1 cells, although the incorporation of [3H]glucosamine into total extracts and NCAM was almost the same. In contrast, when cells were pulse labeled with [35S]methionine followed by a 1 h chase, there was not such a great difference in PSA-carrying protein synthesis between K1 and Lec13 cells. However, during a prolonged chase period, PSA-carrying proteins rapidly decreased in Lec13 cells, whereas those in K1 cells did not change. The degradation of PSA-carrying glycoproteins in Lec13 cells was partly prevented when the cells were grown in fucose-containing medium. Therefore, fucose modification of core N-glycans may affect the efficient expression of PSAs through the intracellular stability of PSA-carrying glycoproteins.     

Characterization of infectious aerosols in health care facilities: an aid to effective engineering controls and preventive strategies

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.     

The PEL1 gene (renamed PGS1) encodes the phosphatidylglycero-phosphate synthase of Saccharomyces cerevisiae

Phosphatidylglycerophosphate (PG-P) synthase catalyzes the synthesis of PG-P from CDP-diacylglycerol and sn-glycerol 3-phosphate and functions as the committed and rate-limiting step in the biosynthesis of cardiolipin (CL). In eukaryotic cells, CL is found predominantly in the inner mitochondrial membrane and is generally thought to be an essential component of many mitochondrial functions. We have determined that the PEL1 gene (now renamed PGS1), previously proposed to encode a second phosphatidylserine synthase of yeast (Janitor, M., Jarosch, E., Schweyen, R. J., and Subik, J. (1995) Yeast 13, 1223-1231), in fact encodes a PG-P synthase of Saccharomyces cerevisiae. Overexpression of the PGS1 gene product under the inducible GAL1 promoter resulted in a 14-fold increase in in vitro PG-P synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast did not lead to a loss of viability but did result in a dependence on a fermentable carbon source for growth, a temperature sensitivity for growth, and a petite lethal phenotype. The pgs1 null mutant exhibited no detectable in vitro PG-P synthase activity and no detectable CL or phosphatidylglycerol (PG); significant CL synthase activity was still present. The growth arrest phenotype and lack of PG-P synthase activity of a pgsA null allele of Escherichia coli was corrected by an N-terminal truncated derivative of the yeast PG-P synthase. These results unequivocally demonstrate that the PGS1 gene encodes the major PG-P synthase of yeast and that neither PG nor CL are absolutely essential for cell viability but may be important for normal mitochondrial function.     

Glycosylation defect in Lec1 Chinese hamster ovary mutant is due to a point mutation in N-acetylglucosaminyltransferase I gene

The Lec1 Chinese hamster ovary (CHO) mutant is a leuco-phytohemagglutinin resistant cell line unable to synthesize complex and hybrid N-glycans due to the lack of N-acetylglucosaminyltransferase I (GnTI) activity. Here we have identified the lec1 mutation. Using specific antibodies to GnTI we demonstrate that Lec1 cells synthesize an inactive GnTI protein identical in size to the wild-type CHO enzyme. We have cloned and sequenced the gene coding GnTI from parental CHO and Lec1 mutant cells. Comparison of GnTI sequences detected three mutations within the luminal domain of Lec1 GnTI, each resulting in an amino acid substitution. The effect of each mutation on enzyme activity was analyzed by site-directed mutagenesis of wild-type rabbit GnTI and transient expression in COS cells. One of the three mutations (Cys123 --> Arg123) resulted in complete loss of activity, whereas the other two mutations had no apparent effect on enzyme activity. This conclusion was confirmed by expression of GnTI mutants in the GnTI null background of Saccharomyces cerevisiae. Both Lec1 GnTI and the GnTI mutant (Cys123 --> Arg123) are correctly localized to the Golgi apparatus, indicating that the inactive GnTI molecules are sufficiently well folded for efficient transport from the endoplasmic reticulum. These results demonstrate that the lec1 mutation is a point mutation and that Cys123 is a critical residue for GnTI activity.     

Molecular cloning of the hamster CMP-sialic acid transporter

Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-Sia-Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMP-Sia-Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-Sia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (UDP-Gal-Tr). In contrast, sequence similarities to the yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted to a region encoding the two most C-terminally located transmembrane helices. A computer-based structural analysis of CMP-Sia-Tr proposes an eight transmembrane helix model with the N- and C-termini located on the cytosolic side of the Golgi membrane.     

Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.     

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype

Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker alpha-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter.     

Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae

Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the beta-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100-500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b.