A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-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. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-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.

The hydrophilic and acidic N-terminus of the integral membrane enzyme phosphatidylserine synthase is required for efficient membrane insertion.

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.