Multiple Copies ofPBS2,MHP1 orLRE1 Produce Glucanase Resistance and Other Cell Wall Effects inSaccharomyces cerevisiae

Abstract

Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall (1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall (1-6) glucans. Measurements of (1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 disruption strain was laminarinase super-sensitive and supersensitive to K 1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall (1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight (1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.

Osmoregulation and Fungicide Resistance: the Neurospora crassa os-2 Gene Encodes a HOG1 Mitogen-Activated Protein Kinase Homologue

“…Alternatively, the N. crassa os-2 signal transduction pathway may be involved in other cellular processes, such as cell wall and/or cytoskeleton structure or cell cycle progression. In S. cerevisiae, the HOG pathway has been implicated in regulation of cell wall modification (15) and cytoskeleton reorganization (4) in response to hyperosmotic stresses. The STY1 (HOG1 homologue) MAP kinase pathway of Schizosaccharomyces pombe regulates responses to multiple stresses in addition to hyperosmolarity (11,33) and has roles in cell cycle control and sexual reproduction (31,32).…”

Calcofluor Antifungal Action Depends on Chitin and a Functional High-Osmolarity Glycerol Response (HOG) Pathway: Evidence for a Physiological Role of the Saccharomyces cerevisiae HOG Pathway under Noninducing Conditions

“…Plasmid pCTT1-18/7x (35) was integrated into the chromosome after linearization with NcoI. Plasmids pLJ1 (pRS316::PBS2) and pLJ2 (pRS426::PBS2) were obtained by direct cloning of PBS2 as a SpeI/SacI fragment obtained from plasmid L42 (16). pLJ3 (pRS316::PBS2-14) and pLJ4 (pRS426::PBS2-14) contain a mutated copy of PBS2 generated by site-directed mutagenesis.…”

“…This regulation is translated into significant changes in ␤(1-6)glucan contents, depending on the deletion or overexpression of the PBS2 gene (15). Similarly, deletion or overexpression of this gene leads to significant differences in ␤(1-3)glucan synthase and ␤(1-3)glucan levels through an unknown mechanism (16). This involvement of the HOG pathway in cell wall architecture together with the activation of the PKC pathway under hypo-osmotic conditions (10) suggests an interconnection between these two signaling pathways.…”

Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-Mannosyltransferase I

“…Because Pbn1p has an additional 164-amino acid region containing a coiled-coil structure at the N terminus compared with mammalian PIG-X, it may be a dual-functional protein. Another report described that yeast cells carrying multiple copies of both PBN1 and LRE1 (laminarinase resistance 1) showed resistance to the cell wall ␤1,3-glucan degrading enzyme, although the cells carrying either gene showed no significant alterations (Lai et al, 1997). These observations may suggest that Pbn1p serves as a rate limiting factor for GPI-anchor biosynthesis and interacts with several molecules other than Gpi14p.…”