In vivo interaction between TWD1 and AtMRP1. (A) TWD1-HA overlaps with vacuolar and plasma membrane fractions in a continuous sucrose gradient. Arabidopsis microsomal fractions from plants expressing TWD1-HA were separated by linear sucrose gradient centrifugation and equal amounts of protein of selected fractions were probed with anti-HA and anti-MRP1. Origin of immunopositive fractions was ascertained by Western blotting using antisera against the marker proteins vacuolar PPase, ER localized BIP and the plasma membrane-bound P-type H+-ATPase (). (B) Immunoprecipitation of a TWD1-AtMRP1 complex. Microsomal membranes from Arabidopsis wild-type (lanes 1-5) or TWD-HA plants (lanes 6-9) were cross-linked with DTBP, solubilized using 0.1% Brij 35 and 0.05% CHAPS and immunoprecipitated using an anti-AtMRP1 or anti-HA affinity matrix, respectively. Immunoprecipitated proteins were separated by 7.5% (lanes 1, 2, and 5), 12.5% (lanes 3, 4, 6, and 7) or 4-12% PAGE (lanes 8 and 9) in the presence (+) or absence of DTT (-) and probed with anti-AtMRP1 (lanes 1-2, 8-9), anti-TWD1 (lanes 3-5), and anti-HA (lanes 6-9), respectively. As negative controls, unspecific binding of proteins to empty protein G was monitored (lanes 2 and 4). Note that the size difference of the coprecipitated wild-type TWD1 in lane 3 having a slightly smaller weight than the HA-epitope-tagged TWD1 (lane 6) is due to the lack of the HA-epitope. Molecular size markers on the left and right correspond to lanes 1-2, and 5 and lanes 3-4, 6-7, respectively; positions of TWD1 and AtMRP1 are indicated.