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J. Biol. Chem., Vol. 283, Issue 27, 18553-18565, July 4, 2008

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Disulfide Bond Structure and Domain Organization of Yeast β(1,3)-Glucanosyltransferases Involved in Cell Wall Biogenesis^*

Laura Popolo¹, Enrico Ragni², Cristina Carotti, Oscar Palomares, Ronald Aardema^¶, Jaap Willem Back^¶, Henk L. Dekker^¶, Leo J. de Koning^¶, Luitzen de Jong^¶, and Chris G. de Koster^¶

From the Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, 20133 Milano, Italy, the Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid, Spain, and ^¶Biomolecular Mass Spectrometry Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands

The Gel/Gas/Phr family of fungal β(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component β(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.

Received for publication, February 26, 2008 , and in revised form, April 24, 2008.

^* This work was supported in part by National P.R.I.N. Grant 2005 (to L. P.) and by the European RTN Project 512481 "CanTrain" (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

² Recipient of Fondo Sociale Europeo Fellowship 415438.

¹ To whom correspondence should be addressed. Tel.: 39-2-50314919; Fax: 39-2-50314895; E-mail: Laura.Popolo{at}unimi.it.

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