Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall β‐1,3‐glucan

Abstract

Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by β-mercaptoethanol (β-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys 214 -12aa-Cys 227 -COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys 130 Ser or Cys 197 Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1 and gsc2/fks2 cells, defective in β-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by β-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the β-1,3-glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2-unprocessed Pir4 form. Pir4 failed to be incorporated in alkali-sensitive linkages involving β-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the β-1,3-glucan.

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

“…Although the PIR genes are not essential, strains multiply deleted for PIR1 through PIR4 display additive defects in growth rate, morphology, and sensitivity to cell wall stress agents (Mrsa and Tanner 1999). The Pir proteins are attached directly to b-1,3-glucan chains ( Figure 1) through a linkage that involves their repeat sequences, DGQFQ [where F is any hydrophobic residue (Castillo et al 2003)]. The glucan chain is linked to the protein through the g-carboxyl group of a Glu residue evidently produced through a transglutaminase-type reaction that converts the first Gln residue in the repeat sequence to Glu (Ecker et al 2006).…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Deletion of all PIR sequences from Pir1 and Pir4 leads to release of these proteins from the cells (Castillo et al 2003;Sumita et al 2005), indicating that the repeats are necessary for wall association. The more repeats, the stronger the binding: deletion of increasing numbers of Pir1's repeats led to release of increasing amounts of Pir1 into the medium (Sumita et al 2005).…”

Systematic identification in silico of covalently bound cell wall proteins and analysis of protein-polysaccharide linkages of the human pathogen Candida glabrata

“…CgPir1-4p have all of the above-described Pir protein features, but CgPir5p has no Kex2p cleavage site, no internal repeats, and a very low pI value compared to CgPir1-4p (Table 2). Since studies with mutated versions of ScPir4p/Cis3p indicated that the presence of the glutamine-rich repetitive sequence is required for its covalent attachment to wall carbohydrates (Castillo et al, 2003), it is uncertain whether CgPir5p can be covalently bound to the cell wall.…”

“…Interestingly, in the C-terminal region of all three GPI-modified CgCwp1 proteins a short sequence similar to the internal repeat sequences of Pir proteins is present. These internal repeats have been shown to be essential for covalent coupling of ScPir4 to the cell wall glycan network (Castillo et al, 2003). Such a sequence is also present in ScCwp1, which has been shown to become linked to the cell wall both through GPI modification and in a mild-alkalisensitive manner (Kapteyn et al, 2001).…”