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.

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200 -300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of ␣-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.Protein glycosylation in eukaryotic cells is thought to play an essential role in many processes such as protein folding and transport, maintenance of protein and cell structure, and cell recognition and adhesion, as well as other functions. From the several types of protein glycosylation, N-glycosylation has received a great deal of attention not only because of its high frequency but also because several biochemical steps involved in this biosynthetic process are shared by yeast and humans, an indication that they have been conserved throughout evolution. These conserved steps occur in the membrane (i) or the lumen (ii and iii) of the ER 1 and belong to three groups: (i) assembly of the precursor oligosaccharide, GlnNAc 2 -Man 9 Glc 3 on a lipid carrier (dolichol-PP), (ii) transfer of the oligosaccharide to the nascent or recently synthesized protein acceptor, and (iii) trimming of the three glucoses and one mannose (for recent reviews, see Refs. 1 and 2).However, once the glycoprotein leaves the ER, biochemical modification by trimming and/or addition of new sugars varies enormously between species and even between individual proteins of the same cell. This suggests that individual proteins carry the precise information for the final carbohydrate composition. In Saccharomyces cerevisiae, some of the protein-attached oligosaccharides leaving the ER (GlcNAc 2 -Man 8 ) are poorly elongated with up to 13-14 mannoses (coretype), whereas many others are further elongated by the addition of an outer chain of up to 200 mannose residues in the Golgi apparatus (mannan-type), a process commonly known as hyperglycosylation. The outer chain consists of a backbone of ␣-1,6-mannoses with ␣-1,2 branches that are decorated with terminal ␣-1,3-mannose residues (1, 3). The biosynthesis of this complex is carried out...