Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing.

Abstract

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.