The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b5 and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-fl-glucosaminidase, ~3-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine-and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall. Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation. On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.Yeast flocculation is an asexual, calcium-dependent, and reversible aggregation of cells into flocs. This phenomenon is thought to involve cell surface components. It is widely accepted that it results from a lectin-like interaction between a cell wall sugar-binding protein and cell surface mannan (22,30).Yeast flocculation is under genetic control, and three dominant flocculation genes have been defined by classical genetics, FLO1, FLO5, and FLO8 (13). The FLO1 gene is the gene that has been most studied, and it has been cloned and sequenced by different groups (3,35,39). Systematic sequencing of the yeast genome has recently led to the identification of other open reading frames which are homologous with the FLO1 gene (5, 14). It is likely that two of them correspond to the already genetically defined FLO5 and FLO8 genes, while others are new putative flocculation genes (34).The predicted Flo1 protein is a large, 1,536-amino-acid (aa) serine-and threonine-rich polypeptide which contains numerous repeated sequences and a potential signal peptide for secretion (39). In addition, the Flo1 product possesses hydrophobic C-terminal sequences which are characteristic of signals for glycosyl phosphatidylinositol (GPI) anchor addition (23). These features are consistent with a cell surface localization of the Flo1 protein. We have reported immunological evidence of this type of localization with the Flo1 protein in a previous work (4). A Flo1 homologous protein has also ...

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix‐localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single‐stranded (ss) DNA‐dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5′ to 3′ direction with respect to the ss DNA on which it binds first.

We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977-986] encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1-phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent. On glucose medium, the 40-fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth. In contrast, on galactose, the 40-fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3-5-fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1-phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5-10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10-fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall beta-glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism.