Disulfide bond structure and domain organization of yeast beta(1,3)-glucanosyltransferases involved in cell wall biogenesis.
The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component beta(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.
Popolo
Laura
L
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, 20133 Milano, Italy. Laura.Popolo@unimi.it
Ragni
Enrico
E
Carotti
Cristina
C
Palomares
Oscar
O
Aardema
Ronald
R
Back
Jaap Willem
JW
Dekker
Henk L
HL
de Koning
Leo J
LJ
de Jong
Luitzen
L
de Koster
Chris G
CG
D019943:Amino Acid Substitution; D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; C066709:GAS1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D003545:Cysteine