Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

Abstract

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. Fungal cell walls are rigid albeit dynamic, complex structures, withstanding intracellular osmotic pressures of more than 10 bar (1). In Saccharomyces cerevisiae the cell wall amounts to about 20% of the total dry weight of the cell. Besides a small amount of chitin, it mainly consists of ␤-glucans and mannoproteins. The actual protein content comes to about 10% of the wall by weight (2). Almost all the proteins are highly Oand N-mannosylated (2-5). Cell wall studies in the past ten to fifteen years have focused on the various cell wall proteins, since the information concerning their function is expected to help understand the biogenesis as well as the dynamic rebuilding and dissolving of this highly intricate, extracellular organelle during budding and mating.By now, more than 30 cell wall proteins have been identified (6 -9). Some of them are solubilized from the cell wall by SDS under reducing conditions and are therefore called soluble cell wall proteins (10), however, most of them are covalently attached to the glucan layer and can be released from SDS-extracted cell walls by ␤-glucanases (11, 12). Two modes of covalent linkage can be distinguished. In the first case the cell wall protein is synthesized as GPI 5 (glycosylphosphatidylinositol) modified intermediate. These are trans-mannosylated by hydrolyzing the oligomannosyl moiety of the GPI lipid anchor and by a subsequent transfer to ␤-1,6-glucan (12-16). This group of covalently attached cell wall proteins, called GPI-Cwp, are released from the wall both by ␤-1,6-and by ␤-1,3-glucanases, due to the attachment of GPI-Cwps to ␤-1,3-glucan via a short ␤-1,6-glucan bridge (4, 12). The second group of covalently linked cell wall proteins are the so-called Pir proteins, named originally according to three genes coding for putative proteins with internal repeats (Pir) (17) and subsequently identified as covalently linked cell wall proteins Ccw1, -2, -3, and -4p (8,18,19). For S. cerevisiae five PIR genes are known (Table 1) coding for proteins with varying numbers of repetitive units (1 to 10). Pir proteins do not contain a C-terminal GPI-addition signal, they are all processed by Kex2 protease, and they are released from intact cells by very mild alkaline treatment (30 mM NaOH, 12 h, 4°C) (8). Pir proteins are attached directly to ␤-1,3-glucan (20) and can therefore be released from S. cerevisiae by the corresponding ␤-1,3-glucanase, not however by ␤-1,6-glucanase (4). The actual alkali labile linkage between the protein moiety and ␤-1,3-glucan is not known. Pir proteins known to be involved in various biological phenomena are not essential for viability; a quadruple mutant, however, possesses a severe growth phenotype (21). Pir2/Ccw7/Hsp150p is strongly induced by heat stress and ni...

Binding assay for incorporation of alkali‐extractable proteins in the Saccharomyces cerevisiae cell wall

“…It was reported previously that Scw4p is a protein non-covalently attached to glucan (Cappellaro et al, 1998), but even after extensive extraction of cell walls with hot SDS, a portion of the protein remained and could only be extracted from the walls by mild alkali. Such a result is surprising, since Scw4p does not contain the Pir-repetitive sequence shown to be an absolute requirement for incorporation of Pir proteins by an alkali-sensitive linkage (Ecker et al, 2006). However, this is in accordance with the recent findings of Yin et al (2005), who also identified Scw4p among alkaliextractable cell wall proteins, using tandem mass spectrometry of peptides released by proteolytic digestion of SDS-treated S. cerevisiae cell walls.…”

“…Plasmids containing CCW5 with exchanged specific amino acids pCCW5-HA used in this work (CCW5 plasmid containing haemagglutinin epitope at the C-terminus) was prepared by PCR as already described by Ecker et al (2006), as well as pCCW5 plasmids containing CCW5 with exchanged specific amino acids [pCCW5-(Q69A)-HA, pCCW5- …”

“…They are usually extracted from the wall by either β-1,6-glucanases or β-1,3-glucanases, although restricted proteolysis has also been reported (Shimoi et al, 1995). Finally, a family of sequencerelated proteins called Pir proteins (proteins with internal repeats) are attached to β-1,3-glucan by a newly described type of alkali-labile ester linkage between the γ -carboxyl group of the specific glutamic acid residue within the protein repetitive sequence and the hydroxyl group of glucose (Ecker et al, 2006). Four Pir proteins were extracted from the wall by a mild alkali treatment and were shown to share several common structural features, the 260 R. Teparić, I. Stuparević, V. Mrša most prominent being the repetitive sequence at the N-terminus of all four proteins (Mrša et al, 1997).…”

“…Deletion analysis of Pir proteins showed that the repeating sequence is absolutely required for binding to glucan, and mutagenesis experiments revealed that three glutamines and one aspartic acid within the sequence were essential. Glutamine at position 74 was identified as the linking site (Ecker et al, 2006). It is not known how this reaction takes place and which enzyme is responsible for the formation of the ester linkage, since no cell wall protein mutant described so far, single or multiple, has been shown to release Pir proteins into the medium.…”

“…It is not known how this reaction takes place and which enzyme is responsible for the formation of the ester linkage, since no cell wall protein mutant described so far, single or multiple, has been shown to release Pir proteins into the medium. Moreover, a possibility of autocatalytic incorporation of these proteins into the wall has been discussed (Ecker et al, 2006).…”