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 sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined as FLO5 (Bidard et al., 1994) has revealed that it was a FLO1 gene. The FLO1 gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy. The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein. A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level. A gene disruption experiment has shown that FLO5 strain STX 347-1D contains at least two flocculation genes of the FLO1 type but that they are supposed to be inactive and do not contribute to its flocculation. However, enzyme-linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of the FLO5 strain STX 347-1D is antigenically related to Flo1p. A deletion analysis of the 5' region of the FLO1 gene has shown that the expression is submitted to controls which depend on the genetic background of the strain.

In Saccharomyces cerevisiae, L-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased L-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), L-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: V max ‫؍‬ 8.7 nmol/mg/min; K m ‫؍‬ 1.6 mM) and some simple diffusion of the undissociated L-malic acid (K d ‫؍‬ 0.057 min ؊1 ). As total L-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. L-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ⌬pH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to L-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited L-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated L-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport.Previous studies have provided evidence that L-malic acid is metabolized by Saccharomyces cerevisiae only in the presence of an assimilable carbon source (16,24,25). Exogenous L-malic acid (3 g/liter) is always consumed to a limited extent (10 to 20%), and the amount of degraded malate depends on the strain and culture conditions. This incomplete consumption of L-malic acid may be due to limited malate uptake and inefficiency of the enzyme systems involved in metabolism of the acid. Indeed, it has been reported that the transport of Lmalate is not carrier mediated in S. cerevisiae; the undissociated form of the acid slowly enters the cell by simple diffusion (28). During fermentation in the presence of malate, intracellular malate concentration in this yeast (close to 1 mM) is therefore lower than that in yeasts having a carrier protein for L-malate and able to metabolize L-malic acid completely (10 to 15 mM in Saccharomyces bailii) (20). The constitutive L-malic enzyme, thought to be responsible for the anaerobic metabolism of L-malate in S. cerevisiae, has a high K m for the substrate (50 mM) (16,17). Given the low levels of intracellular malate, the malic enzyme seems to function to a limited extent. Anaerobically grown S. cerevisiae cells also contain a ma...

The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation. We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence. Our data show that they are not permanently present at the cell surface but that their amount increases during growth. With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level. Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins. The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes. Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds. Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells. Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins. Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment.