improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol A24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDP-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol A24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.Most of the enzymes involved in cellular phospholipid biosynthesis are membrane associated. In mammalian cells, the majority of phospholipids is synthesized in the endoplasmic reticulum (14). Phospholipids specifically required for mitochondrial function (cardiolipin and its precursor phosphatidylglycerol) as well as phosphatidylethanolamine (via decarboxylation of phosphatidylserine) are synthesized in mitochondrial membranes (11).In previous studies, several enzymes of phospholipid biosynthesis of the yeast Saccharomyces cerevisiae (10,26), namely glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine synthase, and phosphatidylinositol synthase, were detected both in the microsomal fraction and in the outer mitochondrial membrane. These observations were based mainly on the separation of subcellular membranes by differential centrifugation and on commonly used marker enzymes for the respective fractions. Motivated by our interest in the mechanisms of lipid flow and membrane assembly in yeasts and by conflicting data concerning the subcellular targeting of phosphatidylserine synthase (38), we reinvestigated the subcellular distribution of lipid-synthesizing enzymes by employing recently developed or improved fractionation procedures for mitochondrial and microsomal membranes, the nuclear membrane (24), the plasma membrane (37) Yeast subcellular membranes were also characterized with respect to their protein-to-lipid ratio, their content of ergosterol and ergosteryl esters, and their pattern of individual glycerophospholipids. Measurements of fluorescence anisotropy revealed significant difference...

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In Saccharomyces cerevisiae, the membrane-associated enzyme phosphatidylserine synthase (EC 2.7.8.8) is present in the mitochondria and the endoplasmic reticulum. The enzyme from both membrane fractions reacted with antiserum raised against a hybrid protein expressed from a TRPE-CHO1 fusion gene in Escherichia coli and was absent in a cho1 null mutant, strongly suggesting that both the mitochondrial and microsomal forms of phosphatidylserine synthase are the products of the CHO1 gene. The highest degree of purification of enzymatically active protein was 380- and 420-fold from the mitochondrial and the microsomal compartments, respectively. In both cases, the enzymatically active and immunoreactive material comigrated with a protein band of 30,000 apparent molecular weight. In the absence of protease inhibitors during the preparation of membranes, the enzyme underwent degradation to an enzymatically active protein of 23,000 apparent molecular weight.

The product of the yeast CHO 1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1.2 kb fragment containing the regulatory and structural components of the CHO 1 gene was sequenced. Transcription initiation in wild-type cells was found to occur between -1 and -15 relative to the first ATG of a large open reading frame capable of encoding a 30,804 molecular weight protein. This translation initiation site was active in vivo and in vitro in a heterologous system. In both cases it supported production of a protein of approximately 30,000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22,400 molecular weight. A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22,000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a cho 1 null-mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast. Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.

The Eurachem-CITAC Guide Quantifying Uncertainty in Analytical Measurement was put into practice in a public laboratory devoted to environmental analytical measurements. In doing so due regard was given to the provisions of ISO 17025 and an attempt was made to base the entire estimation of measurement uncertainty on available data from the literature or from previously performed validation studies. Most environmental analytical procedures laid down in national or international standards are the result of cooperative efforts and put into effect as part of a compromise between all parties involved, public and private, that also encompasses environmental standards and statutory limits. Central to many procedures is the focus on the measurement of environmental effects rather than on individual chemical species. In this situation it is particularly important to understand the measurement process well enough to produce a realistic uncertainty statement. Environmental analytical methods will be examined as far as necessary, but reference will also be made to analytical methods in general and to physical measurement methods where appropriate. This paper describes ways and means of quantifying uncertainty for frequently practised methods of environmental analysis. It will be shown that operationally defined measurands are no obstacle to the estimation process as described in the Eurachem/CITAC Guide if it is accepted that the dominating component of uncertainty comes from the actual practice of the method as a reproducibility standard deviation.