CONSTRUCTION OF A DIRECTORY OF TOBACCO PLASMA-MEMBRANE PROTEINS BY COMBINED 2-DIMENSIONAL GEL-ELECTROPHORESIS AND PROTEIN SEQUENCING

The polypeptide pattern of the plasma membrane from tobacco was studie d by two-dimensional gel electrophoresis, When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate - polyacrylamid e gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spo ts were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient io assess physiologica l effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approxi mately 600 polypeptides, corresponding to ca. 80% of the total populat ion expected, were resolved, This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 lu 45 kDa) and acidic (5,2 < pi < 6,1) polypeptides , After sample application by in-gel rehydration, large amounts of pla sma membrane protein (between 5 mg and 10 mg protein) were analyzed us ing IPG-IEF, and N-terminal protein sequencing was performed for polyp eptides collected from one gel, Internal protein sequences mere also o btained, Nearly all protein sequences corresponded to unidentified pro teins but several of them matched translated sequences from unidentifi ed plant expressed sequence tags (ESTs), It is concluded that the comb ined use of IPG-IEF gels and in-gel rehydration allows, in II-re case of giant membrane protein, both analytical and micropreparative separa tions with an efficiency comparable to that demonstrated for soluble p roteins,: 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.