Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins.
We have carried out a screen of 622 deletion strains generated during the EUROFAN B0 project to identify non-essential genes related to the mannosylphosphate content of the cell wall. By examining the affinity of the deletants for the cationic dye alcian blue and the ion exchanger QAE-Sephadex, we have selected 50 strains. On the basis on their reactivity (blue colour intensity) in the alcian blue assay, mutants with a lower phosphate content than wild-type cells were then arranged in groups defined by previously characterized mutants, as follows: group I (mnn6), group II (between mnn6 and mnn9) and group III (mnn9). Similarly, strains that behaved like mnn1 (i.e. a blue colour deeper than wild-type) were included in group VI. To confirm the association between the phenotype and a specific mutation, strains were complemented with clones or subjected to tetrad analysis. Selected strains were further tested for extracellular invertase and exoglucanase. Within groups I, II and III, we found some genes known to be involved in oligosaccharide biosynthesis (ALG9, ALG12, HOC1), secretion (BRE5, COD4/COG5, VPS53), transcription (YOL072w/THP1, ELP2, STB1, SNF11), cell polarity (SEP7, RDG1), mitochondrial function (YFH1), cell metabolism, as well as orphan genes. Within group VI, we found genes involved in environmentally regulated transduction pathways (PAL2 and RIM20) as well as others with miscellaneous or unknown functions. We conclude that mannosylphosphorylation is severely impaired in some deletants deficient in specific glycosylation/secretion processes, but many other different pathways may also modulate the amount of mannosylphosphate in the cell wall.
Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.
Raúl
R
Conde
Guadalupe
G
Pablo
Rosario
R
Cueva
Germán
G
Larriba
D000423:Alcian Blue / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal / Q000502:physiology; D043326:Glucan 1,3-beta-Glucosidase / Q000378:metabolism; D008360:Mannosephosphates / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000378:metabolism
D004396:Coloring Agents; D005656:Fungal Proteins; D008360:Mannosephosphates; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; C025363:mannoproteins; D043324:beta-Fructofuranosidase; C468487:EXG1 protein, S cerevisiae; D043326:Glucan 1,3-beta-Glucosidase; D000423:Alcian Blue