Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion.

The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Sigma1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Sigma1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae.

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Sigma1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

Development of pseudohyphae by embedded haploid and diploid yeast.

Diploid strains of S. cerevisiae are known to develop pseudohyphae in response to starvation for nitrogen. We report that both haploid and diploid yeast grow in a filamentous form when embedded in solid media. This is not a response to starvation, since yeast grown on rich media and overlaid with rich agar grow within the agar as pseudohyphae. While we find that the only element of diploidy required for formation of pseudohyphae in response to nitrogen starvation is the a1/alpha 2 repressor, pseudohyphal development by embedded cells does not require a1/alpha 2. Deletion of BUD 5 prevented the formation of pseudohyphae by embedded cells, suggesting that these structures are the result of ordered filament formation rather than agar penetration. Deletion of STE 12 prevented the formation of pseudohyphae by all cell types, showing that the same signal transduction pathway is used by embedded cells as by those responding to nitrogen starvation. Different cell types of yeast thus form filaments in response to several kinds of environmental stimuli.

FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin.

We report the characterization of a gene encoding a novel flocculin related to the STA genes of yeast, which encode secreted glucoamylase. The STA genes comprise sequences that are homologous to the sporulation-specific glucoamylase SGA and to two other sequences, S2 and S1. We find that S2 and S1 are part of a single gene which we have named FLO11. The sequence of FLO11 reveals a 4,104-bp open reading frame on chromosome IX whose predicted product is similar in overall structure to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. An amino-terminal domain containing a signal sequence and a carboxy-terminal domain with homology to GPI (glycosyl-phosphatidyl-inositol) anchor-containing proteins are separated by a central domain containing a highly repeated threonine- and serine-rich sequence. Yeast cells that express FLO11 aggregate in the calcium-dependent process of flocculation. Flocculation is abolished when FLO11 is disrupted. The product of STA1 also is shown to have flocculating activity. When a green fluorescent protein fusion of FLO11 was expressed from the FLO11 promoter on a single-copy plasmid, fluorescence was observed in vivo at the periphery of cells. We propose that FLO11 encodes a flocculin because of its demonstrated role in flocculation, its structural similarity to other members of the FLO gene family, and the cell surface location of its product. FLO11 gene sequences are present in all yeast strains tested, including all standard laboratory strains, unlike the STA genes which are present only in the variant strain Saccharomyces cerevisiae var. diastaticus. FLO11 differs from all other yeast flocculins in that it is located near a centromere rather than a telomere, and its expression is regulated by mating type. Repression of FLO11-dependent flocculation in diploids is conferred by the mating-type repressor al/alpha2.

DNA-protein interactions at the S.cerevisiae alpha 2 operator in vivo.

Two homodimeric proteins, alpha 2 and MCM1, are required to repress transcription of a-cell type specific genes in haploid yeast alpha-cells. In vitro studies by others of the interactions of these proteins with operator DNA have suggested that MCM1 binds to the middle and alpha 2 to the ends of the 31 bp operator. We have previously shown that alpha 2 organizes chromatin structure adjacent to the operator; in the presence of alpha 2 repressor, a precisely positioned nucleosome abuts the operator in both minichromosomes and the genome. We present in vivo footprinting evidence consistent with occupancy of the operator by MCM1 in both a- and alpha-cells and by alpha 2 repressor in alpha-cells. Interestingly, our in vivo results differ from previous in vitro work in detail. In contrast to the broad block of reagent accessibility to DNA by the factors seen in vitro, we find a pattern of strand-specific protection or augmented reactivity in vivo. The in vivo results are consistent with genetic data concerning transcriptional regulation of a-cell specific genes and corroborate the crystallographic data of others.

Binding of yeast a1 and alpha 2 as a heterodimer to the operator DNA of a haploid-specific gene.

The mating-type locus (MAT) encodes several DNA-binding proteins, which determine the three cell types of Saccharomyces cerevisiae: the a and alpha haploid cell types, and the a/alpha diploid cell type. One of the products of MAT, alpha 2, functions in two cell types. In alpha cells, alpha 2 represses the a-specific genes by binding to the operator as a dimer. In a/alpha diploid cells, alpha 2 acts with a1, a product of the other MAT allele, to repress a different set of genes, the haploid-specific genes. Until now, the nature of the interaction between a1 and alpha 2 was not known, although it had been suggested that alpha 2 may form a heterodimer with a1. I show, by using proteins synthesized in vitro, that a1 and alpha 2 bind the operator of a haploid-specific gene as a heterodimer. The ability of alpha 2 to form both homodimers and heterodimers with a1, each with a different DNA-binding specificity, explains the dual regulatory functions of alpha 2. This is the first example of regulation by heterodimerization among homeobox-containing proteins, a class that includes proteins responsible for the specification of segment identity in Drosophila, mammals and other eukaryotes.

Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae.

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

Evolution of the amylase multigene family. YBR/Ki mice express a pancreatic amylase gene which is silent in other strains.

The two isozymes of pancreatic amylase in mouse strain YBR/Ki are encoded by closely linked genes which are independently regulated. We have isolated these two pancreatic amylase genes, Amy-2.1 and Amy-2.2, from a cosmid library of YBR/Ki genomic DNA and compared the nucleotide sequences of coding regions with the amino acid sequences of the protein isozymes. Transcripts of both genes were also isolated from a pancreatic cDNA library and partially sequenced. The results demonstrate that Amy-2.1 encodes the A1 isozyme of YBR/Ki pancreatic amylase, while Amy- 2.2 encodes the insulin-dependent B1 isozyme. Similarities of restriction maps and nucleotide sequences suggest that Amy-2.1 is closely related to the active Amy-2a gene previously isolated from strain A/J (Schibler, U., Pittet, A.-C., Young, R. A., Hagenbüchle, O., Tosi, M., Gellman, S., and Wellauer, P. K. (1982) J. Mol. Biol. 155, 247-266). Expression of Amy-2.2 may be limited to strain YBR/Ki. The inactive Amy-X gene from A/J (Schibler, U., Pittet, A.-C., Young, R. A., Hagenbüchle, O., Tosi, M., Gellman, S., and Wellauer, P. K. (1982) J. Mol. Biol. 155, 247-266) is apparently a null allele of Amy-2.2. An additional amylase gene from YBR/Ki has been identified as a pancreatic amylase pseudogene which diverged between sixteen and thirty-two million years ago. The pancreatic amylase subfamily in strain YBR/Ki thus consists of two active genes and one pseudogene. The low rate of amylase production in YBR/Ki pancreas, relative to that of other inbred strains, can be accounted for by the lower number of gene copies in this strain. Comparison of pancreatic amylase genes from different inbred strains provides evidence for several duplication and deletion events during the recent evolution of this chromosome region.

Independent regulation of nonallelic pancreatic amylase genes in diabetic mice.

Administration of streptozotocin produces a diabetic condition in mice characterized by a specific decrease in amylase synthesis in the pancreas as well as a substantial reduction in amylase mRNA concentration. We have studied this effect in mice of the congenic strains C3H.AmyYBR and C3H.AmyCE with multiple active copies of the pancreatic amylase structural gene. When mice of these strains are treated with streptozotocin, the magnitude of reduction in the synthesis of each amylase isozyme is different. These differences are reflected in the relative activities of isozyme-specific mRNAs in an in vitro translation assay. Administration of insulin results in partial restoration of normal phenotypes. The results provide genetic evidence that individual copies of the amylase structural gene are associated with divergent cis-acting insulin-responsive sequences.