Osamu Kuge

A Mammalian Homolog of the Yeast LCB1 Encodes a Component of Serine Palmitoyltransferase, the Enzyme Catalyzing the First Step in Sphingolipid Synthesis

Serine palmitoyltransferase (SPT; EC2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 andLCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and anLCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT.

Isolation of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylglycerophosphate (PGP) Synthase, Expression of Which Corrects the Mitochondrial Abnormalities of a PGP Synthase-defective Mutant of CHO-K1 Cells

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 namedPGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1protein with a N-terminal extension in Escherichia coliresulted 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.

Phosphatidylserine synthase I and II of mammalian cells.

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through an exchange of free L-serine for the base moiety of pre-existing phospholipids. Studies on PtdSer biosynthesis in Chinese hamster ovary (CHO) cells have suggested that the serine base-exchange is catalyzed by at least two different enzymes; one, named PtdSer synthase I (PSS I), uses phosphatidylcholine (PtdCho) and possibly phosphatidylethanolamine (PtdEtn) as phosphatidyl donors for the serine base-exchange, and the other, named PtdSer synthase II (PSS II), uses PtdEtn but not PtdCho as a phosphatidyl donor. Recently, cDNAs of the PSS I and II have been isolated from CHO-K1 cells. This review will briefly describe the current understanding of PtdSer synthases of mammalian cells, mainly CHO cells.

Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria

CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

Cloning of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylserine Synthase (PSS) II, Overexpression of Which Suppresses the Phosphatidylserine Biosynthetic Defect of a PSS I-lacking Mutant of CHO-K1 Cells*

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free l-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated aspssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssBcDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of thepssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. ThepssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.

The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes

ABSTRACT Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA+ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA+ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange ofl-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a ∼4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssBcells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.

Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells

We have previously shown that a Chinese hamster ovary (CHO) cell mutant defective in phosphatidylserine synthase I recovers the enzyme activity on transfection with a pssA cDNA clone isolated from the parental CHO‐K1. The resultant transfectant, CDT‐1, exhibited about 20‐fold higher specific activity of the enzyme in the membrane fraction than CHO‐K1 cells. Polyclonal antibodies against two peptides of the predicted pssA product cross‐reacted with a membrane protein having an apparent molecular mass of 42 kDa, which was overproduced in CDT‐1 cells. By immunoprecipitation with the antibody, phosphatidylserine synthase I activity as well as the 42‐kDa protein was eliminated from solubilized membrane proteins of CDT‐1 cells. Both the enzyme activity and the 42‐kDa protein of CHO‐K1 cells were enriched in the mitochondria‐associated membrane fraction and the microsome fraction, but neither was enriched in the mitochondria fraction or the cytosol fraction. These results suggest that the pssA gene encodes phosphatidylserine synthase I.

Enhancement of Transport-dependent Decarboxylation of Phosphatidylserine by S100B Protein in Permeabilized Chinese Hamster Ovary Cells

Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His6-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.