Processing of yeast exoglucanase (β‐glucosidase) in a KEX2‐dependent manner

Basco, Ricardo D.; Giménez‐Gallego, Guillermo; Larriba, Germán

doi:10.1016/0014-5793(90)80982-o

Cited by 20 publications

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“…Since only the second oligosaccharide of Exg1p can be elongated (Basco et al , 1994), both hyperglycosylated fractions represent different stages in the elongation of a single oligosaccharide. Finally, Exg1p is synthesized as a protein precursor (pro‐Exg1p) that is cleaved by Kex2p to yield the mature Exg1p (Basco et al , 1990). This precursor elutes as a rather sharp peak in fractions 11–13 from the ion exchange column, but shifts to fraction 21 following deglycosylation with endo H (see Figure 2a, E).…”

Section: Resultsmentioning

confidence: 99%

“…(E) Arrows indicate, from left to right, the precursor and mature forms of Exg1b (column 1) and their endoH‐treated forms, respectively (column 2). Form A contains the tetrapeptide Asn–Lys–Lys–Arg preceding the amino terminus of Exg1p (Basco et al , 1990). (b) Fractions 17–21 (B) and 16–33 (C, D) were concentrated and analysed in SDS–PAGE…”

Section: Resultsmentioning

confidence: 99%

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Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins

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Yeast

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins

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Yeast

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“…A number of actual and putative glucanases and transglucosidases have been identified in S. cerevisiae (3,6,10,44,57). Again, few details regarding their specific functions are known, but it is presumed that the glucanases and transglucosidase are involved in remodeling of the cell wall (59).…”

Section: Discussionmentioning

confidence: 99%

“…The founding members of this family, YPS1 and -2 (17,32), were identified as suppressors of null mutations in KEX2, a trans-Golgi network-localized serine protease that processes secretory proteins such as pro-␣-factor and the exoglucanase Exg1p (3,53). The remaining three yapsins were subsequently identified by sequence homology following completion of the S. cerevisiae genome sequence (47).…”

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Yapsins Are a Family of Aspartyl Proteases Required for Cell Wall Integrity in Saccharomyces cerevisiae

et al. 2005

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The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositollinked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1⌬2⌬ mutant and the quintuple yapsin mutant (5yps⌬) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5yps⌬ and yps1⌬2⌬ mutants have decreased amounts of 1,3-and 1,6-␤-glucan. Although there is decreased incorporation of both 1,3-and 1,6-␤-glucan in the 5yps⌬ mutant in vivo, in vitro specific activity of both 1,3-and 1,6-␤-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5yps⌬. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1⌬ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.The yapsins are a family of five glycosylphosphatidylinositol (GPI)-linked aspartyl proteases (YPS1 to -3, -6, and -7) in Saccharomyces cerevisiae that have homologues in other fungi such as Candida albicans (43), Candida glabrata (16), and Aspergillus oryzae (36). The founding members of this family, YPS1 and -2 (17, 32), were identified as suppressors of null mutations in KEX2, a trans-Golgi network-localized serine protease that processes secretory proteins such as pro-␣-factor and the exoglucanase Exg1p (3, 53). The remaining three yapsins were subsequently identified by sequence homology following completion of the S. cerevisiae genome sequence (47). Like Kex2p, three of the yapsins (Yps1p, -2p, and -3p) cleave proteins and peptides C terminal to basic residues, both in vivo and in vitro (8, 33). Although Kex2p is exquisitely selective for Lys-Arg pairs (53), the yapsins cleave C terminal to monobasic sites containing either Lys or Arg (33, 48). In vivo, Yps1p cleaves human mammalian prohormones such as ACTH and CCK (8) and both Yps1p and Yps2p display secretase-like activity toward ␤-amyloid precursor protein that is expressed in yeast (34,66). To date, however, no yeast substrates have been identified; moreover, the physiologic function of the yapsins in S. cerevisiae has been unclear.Previously reported work from this laboratory showed that the yapsin double nul...

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“…Form A is an endoplasmic reticulum form of Exg1 carrying a 21-amino acid propeptide; it is converted to mature form in the Golgi apparatus by elimination of the propeptide by the Kex2 protease (33,34). Although Exg1a and form A co-eluted in HPLC, their deglycosylated products have quite different retention times.…”

Section: Analysis Of the Exg1 Glycoforms Secreted By S Cerevisiae Exmentioning

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A Search for Hyperglycosylation Signals in Yeast Glycoproteins

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Journal of Biological Chemistry

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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...

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Processing of yeast exoglucanase (β‐glucosidase) in a KEX2‐dependent manner

Cited by 20 publications

References 19 publications

Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins

Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins

Yapsins Are a Family of Aspartyl Proteases Required for Cell Wall Integrity in Saccharomyces cerevisiae

A Search for Hyperglycosylation Signals in Yeast Glycoproteins

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