A mutation in the yeast mitochondrial ribosomal protein Rml2p is associated with a defect in catalase gene expression.

Yeast strains containing a new temperature-sensitive allele of the RML2 gene, encoding a component of the large subunit of the mitochondrial ribosome, display normal growth on acetate, slowed growth on glycerol and an inability to grow on oleic acid. These cells, denoted rml2(fat21), have an apparent inability to induce peroxisomal function, as evidenced by a deficiency in oleic acid induction of beta-oxidation. However, the oleic acid regulation of genes encoding core enzymes of peroxisomal beta-oxidation is normal. In contrast, up-regulation of CTA1 (catalase) mRNA expression and enzyme activity is interrupted. Upon comparison of the induction requirements of catalase and the genes of beta-oxidation, we hypothesized that the rml2(fat21) mutation alters the activity of the transcription factor Adr1p. In support of this hypothesis, over-expression of ADR1 in rml2(fat21) cells restores CTA1 induction. Several assays of mitochondria from rml2(fat21) strains suggest normal mitochondrial function. Thus, the modulation of Adr1p-associated gene regulation is not due to overt mitochondrial dysfunction.

The genetics of fatty acid metabolism in Saccharomyces cerevisiae.

Long-chain fatty acids are a vital metabolic energy source and are building blocks of membrane lipids. The yeast Saccharomyces cerevisiae is a valuable model system for elucidation of gene-function relationships in such eukaryotic processes as fatty acid metabolism. Yeast degrades fatty acids only in the peroxisome, and recently, genes encoding core and auxiliary enzymes of peroxisomal beta-oxidation have been identified. Mechanisms involved in fatty acid induction of gene expression have been described, and novel fatty acid-responsive genes have been discovered via yeast genome analysis. In addition, a number of genes essential for synthesis of the variety of fatty acids in yeast have been cloned. Advances in understanding such processes in S. cerevisiae will provide helpful insights to functional genomics approaches in more complex organisms.

Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin.

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+ -ionophore activation. The time-dependence of annexin V incorporation into the detergent-extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re-associated with the actin filaments when re-polymerization was induced in vitro. Using a bifunctional crosslinking reagent we have identified an 85-kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin-stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F-actin to physiological levels. We also show that GST-annexin V mimics the physiological binding of annexin V to membranes, and that this GST-annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and gamma- but not beta-actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely gamma-actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to gamma-actin in an activation-dependent manner forms a strong association with the platelet cytoskeleton.

Methylene blue based protein solder for vascular anastomoses: an in vitro burst pressure study.

BACKGROUND AND OBJECTIVE: Attempts at sutureless anastomoses have used protein-based solders containing chromophores [Oz et al., J Vasc Surg 1990;11:718; Poppas et al., J Urol 1998150:1052] to enhance the strength of laser anastomoses. Reports have described the use of indocyanine green [Oz et al., Surg Forum 1989;316.], fuschin, and fluorescein isothiocyanate as chromophores [Chuck et al. , Lasers Surg Med 1989;9:471; Vance et al., Lasers Med Sci 1988;3:219]. Methylene blue (MB) is a chromophore with absorption peaks in the 600-700 nm region whose use has not been reported in laser-assisted vascular anastomoses. Therefore, we set out to produce and characterise a MB-containing protein solder. The absorption and burst pressure characteristics have been investigated and described as well as a brief review of the chemical and biological properties of MB. STUDY DESIGN/MATERIALS AND METHODS: The MB and porcine serum albumin (PSA)-based solder was produced and used to form end-to-end anastomoses in porcine splenic arteries. The solder was activated using a laser diode emitting at 670 nm. The burst pressures of the anastomoses were tested, and the results analysed as a function of MB concentration and absorption. In addition, the relationship between MB concentration and absorption was examined. RESULTS: A dose-response relationship was found between the measured absorption of the solder and the burst pressure of the anastomoses formed. Burst pressures exceeding physiological levels were found. Changes in MB concentration revealed a marked negative deviation from Beer's law at 670 nm, owing to the monomer-dimer-trimer equilibria. CONCLUSION: PSA with MB solder is able to form high-quality end-to-end anastomoses, with immediate burst pressure profiles similar to those previously described for sutured [Quigley et al., Microsurgery 1985;6:229], lasered [Quigley et al., Microsurgery 1985;6:229], and soldered anastomoses [Small et al., J Clin Laser Med Surg 1997;15:205]. The relationship between burst pressure strength and chromophore absorption is discussed.

Investigation of the relocation of cytosolic phospholipase A2 and annexin V in activated platelets.

Cytosolic phospholipase A(2) is a Ca(2+)-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A(2). Annexin V is a Ca(2+)-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A(2). Here, we have studied the association of cytosolic phospholipase A(2) and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A(2) and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A(2) to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A(2) or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A(2) remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A(2) with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A(2) can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A(2) copurify annexin V and cytosolic phospholipase A(2) from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A(2) and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A(2) and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A(2) into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.

A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast.

Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell. These studies assess the internalization, or retrograde transport, and metabolism of phosphatidic acid in yeast using a fluorescent analog. An analog of phosphatidic acid fluorescently labeled at the sn-2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (NBD-phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles. Transport and metabolism of the NBD-phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods. Primary metabolites of the NBD-phosphatidic acid in yeast were found to be NBD-diacylgycerol and NBD-phosphatidylinositol. Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD-phosphatidic acid to NBD-diacylglycerol is not a pre-requisite for internalization in yeast. Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6-4 mutation, which interrupts endocytosis, has an affect. Thus, internalization of NBD-phosphatidic acid apparently occurs via a non-endocytic route. These characteristics of retrograde transport of NBD-phosphatidic acid in yeast differ significantly from transport of other NBD-phospholipids in yeast as well as NBD-phosphatidic acid transport in mammalian fibroblasts.

Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets.

Immunocytochemical studies demonstrate that annexin V relocates to the plasma membranes of intact stimulated blood platelets. Anti-annexin V antibodies label the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed, glass-activated platelets and colocalize with actin. In addition, crosslinking experiments using detergent-solubilized membranes of activated platelets have identified an 85-kDa complex containing annexin V. The 85-kDa complex is also recognized by antibodies against actin, suggesting that annexin V interacts with actin. In addition, annexin V was found to associate with filamentous actin in the presence of millimolar Ca(2+). Annexin V was also shown by immunofluorescence microscopy to be associated with platelet cytoskeletons, colocalizing with actin in the presence of micromolar Ca(2+). These findings provide the first evidence for annexin V binding to the plasma membrane and to the actin-based cytoskeleton in activated platelets and indicate that annexin V may function in both cytoskeletal and membrane domains.

A yeast strain defective in oleic acid utilization has a mutation in the RML2 gene.

The molecular mechanisms of cellular long-chain fatty acid assimilation and its regulation remain unclear. In an attempt to identify essential mediators of these processes, we have isolated mutant strains of the yeast Saccharomyces cerevisiae unable to utilize oleic acid as sole carbon source, while retaining the ability to utilize acetate. These strains are then subjected to several secondary screening assays to identify mutants of interest. Here we describe a mutant (denoted fat21) that, despite a temperature-sensitive inability to utilize oleic acid as sole carbon source, displays no general defect in oleic acid uptake or incorporation of oleic acid into glycerolipids. Oxidation of acetate after growth in acetate medium is increased similarly in the mutant and parent strains. Oleic acid beta-oxidation in acetate grown cells is also comparable between strains. Induction of oleic acid oxidation following exposure to oleic acid is, however, defective in the fat21 mutant. The fat21 mutant allele displays conditional synthetic lethality in combination with a null allele of the OLE1 gene, which encodes Delta9-desaturase and is required for proper mitochondrial segregation. Clones capable of complementing the fat21 defect contained the RML2 gene, encoding a yeast mitochondria ribosomal protein. Segregation analysis and gene replacement experiments demonstrate that RML2 is the gene defective in the fat21 mutant. These observations of a defect in a mitochondrial protein differentially affecting the adaptation to oleic acid and acetate as carbon sources suggest that the phenotype of fat21 is associated with a novel pathway of mitochondrial-nuclear-peroxisomal communication.

A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4-kinase, STT4p, as an essential component in phosphatidylserine metabolism.

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

Localization of annexin VI in the adult and neonatal heart.

Annexins are a major family of intracellular Ca(2+)-binding proteins which have been implicated in a variety of cellular functions. In this paper the authors have used confocal microscopy to compare the distribution of annexin VI in vibratome sections of the rat adult left ventricle and striated muscle of the rat oesophagus. It is shown that in rat cardiac myocytes annexin VI is associated with only the sarcolemma and intercalated discs. In contrast, it is demonstrated that in rat skeletal muscle annexin VI is associated with the sarcoplasmic reticulum, in addition to the plasma membrane, suggesting that annexin VI is regulating different processes in these tissues. Also shown is that in vibratome sections of the neonatal rat left ventricle, annexin VI has a different subcellular location to that observed in the terminally differentiated adult myocyte. In these differentiating neonatal cells annexins VI is also associated with specific subcellular structures. Furthermore, using confocal microscopy of isolated myocytes the authors demonstrate that the association of annexin VI with the sarcolemma is stable even after cells are treated with the intracellular calcium chelator BAPTA-AM, to greatly deplete cytosolic calcium levels. This demonstrates that annexin VI associates tightly with the sarcolemma, and suggests that components in addition to phospholipid are involved in binding annexin VI to the membrane. These results demonstrate that the subcellular location of annexin VI is differentially regulated, and suggest that annexin VI is required for a process or processes characteristic of the sarcolemma, and of the sarcoplasmic reticulum of skeletal but not of heart muscle.

Localization of annexin V in the adult and neonatal heart.

Annexins are a major family of intracellular Ca2+-binding proteins which have been implicated in a variety of cellular functions. Several conflicting reports have been published on the location of annexin V in the heart. In this paper we have used confocal microscopy to demonstrate that annexin V is associated with the sarcolemma and intercalated discs of cardiac myocytes in sections of adult porcine and rat heart. In addition, we have used confocal microscopy of isolated rat myocytes to show that this association is stable even after cells were treated with the intracellular calcium chelator BAPTA-AM, to reduce cytosolic calcium levels to very low levels. This demonstrates that annexin V associates tightly with the sarcolemma and suggests that components in addition to phospholipid are involved in binding annexin V to the membrane. Furthermore, we show that, in sections of the neonatal rat left ventricle, annexin V has a different subcellular location than that observed in the terminally differentiated adult myocyte. In these differentiating neonatal cells, annexin V is also located in the nucleoplasm and at the periphery of the nucleus. These results demonstrate that the subcellular location of annexin V is differentially regulated and suggest that annexin V regulates calcium-dependent processes at both the sarcolemma and the nucleus.

Relocation of annexin V to platelet membranes is a phosphorylation-dependent process.

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5'-[gamma-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.

Fatty acid uptake by Caco-2 human intestinal cells.

The Caco-2 human enterocytic cell line was used to study the kinetics and mechanism of intestinal long chain fatty acid uptake. Initial rates of palmitate (16:0), oleate (18:1), and octanoate (8:0) uptake were determined for adherent cells at greater than 7 days confluence. Uptake of long chain 18:1 and 16:0 by cells grown on coverslips was saturable with an apparent Km of 0.3 microM, but also included a notable diffusive component. Uptake of short chain 8:0, on the other hand, was linear up to 10 microM. Cells grown on permeable Transwell filters were used to study uptake at the apical versus the basolateral membrane. Uptake of long chain (18:1 and 16:0), but not short chain (8:0), fatty acid was saturable at both surfaces with a similar Km of 0.3 microM. In addition, long chain but not short chain fatty acid uptake was competitively inhibitable. Western blot analysis demonstrated that Caco-2 cells express a protein immunoreactive with antibodies to the rat liver plasma membrane fatty acid binding protein (FABPpm), which is thought to be involved in long chain fatty acid transport. Nevertheless, long chain fatty acid uptake was not inhibited by pretreatment of the cells with an FABPpm antibody, nor by pretreatment with two proteases. These data support a saturable component in the transport of long chain but not short chain fatty acids by human intestinal epithelial cells, which may involve an as yet unknown plasma membrane protein.

Synthesis and intracellular transport of aminoglycerophospholipids in permeabilized cells of the yeast, Saccharomyces cerevisiae.

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.

Ca2+ concentration during binding determines the manner in which annexin V binds to membranes.

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with thrombin can induce the association of intracellular annexin V with membranes in two distinct ways. First, in such a way that it can be eluted from the membrane with EGTA and secondly in a manner such that it is tightly bound to the membrane and requires the non-ionic detergent Triton X-100 for its solubilization. We report that exposure of platelets to the calcium ionophore A23187 mimics the relocation induced by stimulation with thrombin. In separate experiments we demonstrate that a calcium ion concentration [Ca2+] of 0.8 microM is sufficient for maximum binding of the EGTA-resistant form to membranes. In contrast a higher [Ca2+] was required to induce maximal binding of the annexin V which could be extracted with EGTA. We demonstrate that following temperature-induced phase separation in Triton X-114, the membrane-associated annexin V partitions predominantly into the aqueous phase. We also show that the isoelectric point of annexin V does not change following membrane association. These observations suggest that a covalent modification, of annexin V itself, is not responsible for its association with the membrane. Millimolar [Ca2+] is required for maximal binding of purified annexin V to phospholipid vesicles. We show that binding to phospholipids can be reversed entirely by subsequent treatment with EGTA. This suggests that the EGTA-resistant form of annexin V is binding to a membrane component other than phosphatidylserine. Annexin V has previously been shown to bind to protein kinase C. Relocation of annexin V to membranes paralleled that of protein kinase C in thrombin-stimulated cells but not in cells treated with A23187, suggesting that these proteins are not functionally linked in platelet activation. Using bifunctional cross-linking reagents we have identified an 85 kDa complex containing annexin V. This may represent an association between annexin V and an annexin V-binding protein with a molecular mass of approximately 50 kDa.

Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae.

Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine. Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R. (1993) J. Biol. Chem. 268, 21416-21424; Clancey, C. J., Chang, S.-C., and Dowhan, W. (1993) J. Biol. Chem. 268, 24580-24590) described the cloning of a yeast structural gene for this enzyme (PSD1) and the creation of the null allele. Based on the phenotype of strains containing a null allele for PSD1 (psd1-delta 1::TRP1) it was hypothesized that yeast have a second phosphatidylserine decarboxylase. The present studies demonstrate the presence of a second enzyme activity (denoted PSD2), which, depending on the method of evaluation, accounts for 4-12% of the total cellular phosphatidylserine decarboxylase activity found in wild type. Recessive mutations resulting in loss of this enzyme activity (denoted psd2) in cells containing the psd1-delta 1::TRP1 null allele also result in ethanolamine auxotrophy. When incubated with [3H]serine these double mutants accumulate label in phosphatidylserine, while very little (< 5%) is converted to phosphatidylethanolamine. In addition, these mutants have a approximately 70% decrease in the amount of total phosphatidylethanolamine even when grown in the presence of exogenous ethanolamine. Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the PSD2 enzyme activity. Unlike the PSD1 activity, the PSD2 enzyme activity does not localize to the mitochondria, but to a low density subcellular compartment with fractionation properties similar to both vacuoles and Golgi.

Phosphatidylserine decarboxylase 2 of Saccharomyces cerevisiáe. Cloning and mapping of the gene, heterologous expression, and creation of the null allele.

The yeast Saccharomyces cerevisiae expresses two phosphatidylserine decarboxylase (PSD) activities which are responsible for conversion of phosphatidylserine to phosphatidylethanolamine, and either enzyme alone is sufficient for normal cellular growth. However, strains containing a PSD1 null allele and a mutation leading to loss of PSD2 activity (psd1-delta 1::TRP1 psd2) are auxotrophic for ethanolamine. This nutritional requirement was utilized to isolate the gene encoding the PSD2 enzyme by complementation. The PSD2 gene encodes a protein of 1138 amino acids with a predicted molecular mass of 130 kDa. The deduced amino acid sequence shows significant identity (34%) to a PSD-like sequence from Clostridium pasteurianum and the yeast PSD1 (19%) at the carboxyl end of the protein. Of particular interest is the presence of a sequence, GGST, which may be involved in post-translational processing and prosthetic group formation similar to other PSD enzymes. The PSD2 amino acid sequence also shows significant homology to the C2 regions of protein kinase C and synaptotagmin. Physical mapping experiments demonstrate that the PSD2 is located on chromosome 7. The PSD2 gene was heterologously expressed by infection of Sf-9 insect cells with recombinant baculovirus, resulting in a 10-fold increase in PSD activity. The null allele of PSD2 was introduced into yeast strains by one-step gene deletion/disruption with a HIS3 marker gene. Strains expressing wild type PSD1 and the psd2-delta 1::HIS3 allele show a small decrease in overall PSD activity, but no noticeable effect upon [3H]serine incorporation into aminophospholipids. Strains containing both the psd1-delta 1::TRP1 and psd2-delta 1::HIS3 null alleles, however, express no detectable PSD activity, are ethanolamine auxotrophs and show a severe deficit in the conversion of [3H]serine-labeled phosphatidylserine to phosphatidylethanolamine. These data indicate that the gene isolated is the structural gene for PSD2 and that the PSD1 and PSD2 enzymes account for all yeast PSD activity.