Two-dimensional gel electrophoresis of membrane proteins from ectomycorrhizal fungi

Abstract

A membrane fraction was isolated from the ectomycorrhizal fungi Pisolithus tinctorius and Cenococcum geophilum and from eucalyptus ectomycorrhizas using differential centrifugation. This fraction contained microsomes free of mitochondrial or nuclear membranes and enriched in endoplasmic reticulum, Golgi, tonoplast and plasma membranes as determined from an analysis of marker enzymes and electron microscopy observations. Four methods of membrane protein solubilisation were assessed on silver-stained 2-dimensional polyacrylamide gels. Gels with limited background staining and streaking and with clearly resolved polypeptides were obtained when P tinctorius and mycorrhizal proteins were extracted with 2% sodium dodecyl sulphate followed by acetone precipitation. On the other hand, the O'Farrell buffer containing urea and Nonidet P-40 was selected for solubilisation of C geophilum membrane proteins. An optimization of solubilisation procedures is therefore required for each fungal species. The procedures described make possible the resolution required for meaningful qualitative and quantitative electrophoretic analysis of membrane proteins from ectomycorrhizal fungi and mycorrhizas.