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.

Amino Acid Sequence↗

PubMedDate not supplied

Expression of a functionally active cardiac fatty acid-binding protein in the yeast, Saccharomyces cerevisiae.

The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind 14C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37 degrees C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added 14C-palmitic acid was significantly reduced both at 30 and 37 degrees C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.

Carbon Radioisotopes↗

PubMedDate not supplied

Effect of cholesterol on vesicle bilayer geometry of choline plasmalogen and comparison with dialkyl-, alkylacyl- and diacyl-glycerophosphocholines.

Small unilamellar vesicles containing alkenylacyl-, alkylacyl-, dialkyl- or diacyl-glycerophosphocholine were prepared by sonication. Their size was determined from the average internal volume after chromatography on Sepharose 2B and from 31P-NMR linewidths. Alkenylacyl glycerophosphocholine (choline plasmalogen) was found to form the largest vesicles. By addition of 30 mol% cholesterol, the size of plasmalogen vesicles, but not of those containing the alkyl and acyl analogue lipids, was significantly increased. The presence of 50 mol% sterol led to highly increased vesicle sizes of alkylacyl, dialkyl and diacyl-glycerophosphocholine. Mixtures of plasmalogens with 50 mol% cholesterol did not form unilamellar vesicles upon sonication. Bilayer thickness and surface area per phospholipid molecule were determined by small angle X-ray scattering and measurement of partial specific volumes. There is little difference between alkenylacyl glycerophosphocholine and the corresponding diacyl-analog, whereas bilayers consisting of dioleoyl glycerophosphocholine are significantly thinner. Correspondingly their molecular surface area is by about 8% larger than that of the mixed-chain diradyl glycerophosphocholine, since the partial molar volumes are similar for all vesicles tested.

Cholesterol↗