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

Yeast - Tập 20 Số 11 - Trang 973-983 - 2003
Luís Castillo1, Ana I. Martínez1, Ana Garcerá1, M. Victoria Elorza1, Eulogio Valentín1, Rafael Sentandreu1
1Departament de Microbiología i Ecología, Facultat de Farmacia, Universtat de Valencia, Avda. Vicent Andrés Estellés s/n, 461000 Burjassot, València, Spain

Tóm tắt

AbstractIdentification 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 (Cys214‐12aa‐Cys227‐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 (Cys130Ser or Cys197Ser) 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 fks1Δ and gsc2/fks2Δ 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. Copyright © 2003 John Wiley & Sons, Ltd.

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Yeast

Tập 20 Số 11

973-983

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