The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA.

Replication of Saccharomyces ribosomal DNA (rDNA) proceeds bidirectionally from origins in a subset of the approximately 150 tandem repeats, but the leftward-moving fork stops when it encounters the replication fork barrier (RFB). The Pif1p helicase and the highly related Rrm3p were rDNA associated in vivo. Both proteins affected rDNA replication but had opposing effects on fork progression. Pif1p helped maintain the RFB. Rrm3p appears to be the replicative helicase for rDNA as it acted catalytically to promote fork progression throughout the rDNA. Loss of Rrm3p increased rDNA breakage and accumulation of rDNA circles, whereas breakage and circles were less common in pif1 cells. These data support a model in which replication fork pausing causes breakage and recombination in the rDNA.

Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum.

Acetyl-CoA carboxylase (ACC1) catalyzes the first and rate limiting step of de novo fatty acid synthesis. Defects in Acc1p were recently correlated with an altered structure/function of the nuclear envelope in yeast. The subcellular distribution of the enzyme was determined in wild-type and mutant cells by cell fractionation and confocal immunofluorescence microscopy. Even though fatty acid synthesis is generally considered to be a cytosolic reaction, we found that Acc1p cofractionated with nuclei and the ER (endoplasmic reticulum) marker BiP/Kar2p. Membrane-bound Acc1p was susceptible to proteinase K digestion and was solubilized by mild salt treatment indicating that it is loosely associated with the cytosolic surface of the nuclear ER membrane. Consistent with these observations, immunofluorescence analysis revealed that Acc1p was distributed in a gradient within the cytoplasm that had its highest concentration around the ER. Possible association of Acc1p with the nuclear pore complexes (NPCs) was investigated in strains that display NPC clustering. Results of these experiments suggest that Acc1p localization is independent of NPC distribution. We propose that association of Acc1p with the cytoplasmic surface of the ER membrane is physiologically relevant to "channel" the enzymatic product of Acc1p, malonyl-CoA, to a putative ER-localized fatty acid chain elongase complex.

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.

Acetyl-CoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism.

We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.