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 beta-1,3-glucan.
Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by beta-mercaptoethanol (beta-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with beta-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 fks1Delta and gsc2/fks2Delta cells, defective in beta-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by beta-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the beta-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 beta-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the beta-1,3-glucan.
Castillo
Luis
L
Departament de Microbiología i Ecología, Facultat de Farmacia, Universtat de Valencia, Avda, Vicent Andrés Estellés s/n, 461000 Burjassot, València, Spain.
Martinez
Ana I
AI
Garcerá
Ana
A
Elorza
M Victoria
MV
Valentín
Eulogio
E
Sentandreu
Rafael
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005936:Glucans / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D047071:beta-Glucans
D005936:Glucans; D029701:Saccharomyces cerevisiae Proteins; D047071:beta-Glucans; C033363:beta-1,3-glucan; D003545:Cysteine