ABSTRACT
In the yeast Saccharomyces cerevisiae, members of the Kre2/Mnt1 protein family have been shown to be α-1,2-mannosyltransferases or α-1,2-mannosylphosphate transferases, utilising an Mn2+-coordinated GDP-mannose as the sugar donor and a variety of mannose derivatives as acceptors. Enzymes in this family are localised to the Golgi apparatus, and have been shown to be involved in both N- and O-linked glycosylation of newly-synthesised proteins, including cell wall glycoproteins. Our knowledge of the nine proteins in this family is however very incomplete at present. Only one family member, Kre2p/Mnt1p, has been studied by structural methods, and three (Ktr4p, Ktr5p, Ktr7p) are completely uncharacterised and remain classified only as putative glycosyltransferases. Here we use in vitro enzyme activity assays to provide experimental confirmation of the predicted glycosyltransferase activity of Ktr4p. Using GDP-mannose as the donor, we observe activity towards the acceptor methyl-α-mannoside, but little or no activity towards mannose or α-1,2-mannobiose. We also present the structure of the lumenal catalytic domain of S. cerevisiae Ktr4p, determined by X-ray crystallography to a resolution of 2.2 Å, and the complex of the enzyme with GDP to 1.9 Å resolution.
Subject(s)
Cell Wall/chemistry , Golgi Apparatus/chemistry , Guanosine Diphosphate Mannose/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Transcription Factors/chemistry , Amino Acid Motifs , Catalysis , Catalytic Domain , Cell Wall/enzymology , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Golgi Apparatus/enzymology , Kinetics , Mannans/chemistry , Methylmannosides/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity , Transcription Factors/geneticsABSTRACT
The membrane protein Erv41p is a major component of COPII-coated vesicles and is thought to play a role in the early secretory pathway in eukaryotic cells. In this study, the full lumenal domain of Erv41p from Saccharomyces cerevisiae (ScErv41p_LD) was recombinantly expressed in Sf9 insect cells and purified by nickel-affinity, ion-exchange and size-exclusion chromatography. ScErv41p_LD crystals were obtained using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.0â Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 49.8, b = 76.9, c = 65.1â Å, α = γ = 90.0, ß = 104.8°.
Subject(s)
Endoplasmic Reticulum , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae , Crystallization , Crystallography, X-Ray , Endoplasmic Reticulum/chemistry , Protein Structure, Tertiary , X-Ray DiffractionABSTRACT
Erv41p is a conserved integral membrane protein that is known to play a role in transport between the endoplasmic reticulum and Golgi apparatus, part of the early secretory pathway of eukaryotes. However, the exact function of the protein is not known, and it shares very low sequence identity with proteins of known structure or function. Here we present the structure of the full lumenal domain of Erv41p from Saccharomyces cerevisiae, determined by X-ray crystallography to a resolution of 2.0Å. The structure reveals the protein to be composed predominantly of two large ß-sheets that form a twisted ß-sandwich. Comparison to structures in the Protein Data Bank shows that the Erv41p lumenal domain displays only limited similarity to ß-sandwich domains of other proteins. Analysis of the surface properties of the protein identifies an extensive patch of negative electrostatic potential on the exposed surface of one of the ß-sheets, which likely forms a binding site for a ligand or interaction partner. A predominantly hydrophobic region close to the membrane interface is identified as a likely site for protein-protein interaction. This structure, the first of Erv41p or any of its homologues, provides a new starting point for studies of the roles of Erv41p and its interaction partners in the eukaryotic secretory pathway.
