A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane

The organization of microtubule arrays in the plant cell cortex involves in teractions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 mem branes, to characterize the protein and isolate the corresponding gene. Scr eening an Arabidopsis cDNA expression library with the antibody 6G5 produce d a partial clone encoding phospholipase D (PLD), and a full-length gene wa s obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospho lipid-metabolizing enzyme and a Ca2+-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Ed man degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the ant ibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubule s, and taxol-incluced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolish ed the labeling. Therefore, p90 is a specialized PLD that associates with m embranes and microtubules, possibly conveying hormonal and environmental si gnals to the microtubule cytoskeleton.