Purification of high-molecular Ltp26/Ltp28 RNP complex weight by TAP chromatography. (A) Mitochondrial extract from Ltp28-TAP transfected cells was sedimented through a glycerol gradient. Each fraction (0.5 mL) was incubated with 20 μL of IgG Sepharose beads and, after extensive washing in 20 mM Tris-HCl, pH 7.6, 60 mM KCl, 10 mM MgCl2, 0.1% NP40, bound material was released from the beads and analyzed for the presence of the endogenous Ltp28 and the Ltp28 TAP fusion protein by Western analysis using anti-p28 antiserum, for the LtREL1 and LtREL2 RNA ligases by adenylation, and for TUTase by Western analysis using anti-TUTase antiserum. The gradient fractions containing the cosedimenting TUTase and RNA ligases are indicated by a line above. (B) IgG purification of Ltp28-associated RNP complex. Mitochondrial extract as in A was subjected to binding to IgG Sepharose and release by cleavage with TEV protease. In a separate experiment, RNase A (0.1 mg/mL) was added during the binding to IgG. The purified “pull down” was fractionated on a glycerol gradient, and the fractions assayed for the RNA ligases and the Ltp26 protein. The fractions showing a depletion of the ligases and Ltp26 are indicated by a line above. (C) Complete TAP purification of mitochondrial extract. This was carried out through the IgG binding with TEV protease release, and calmodulin binding with EGTA release. The purified fraction was subjected to glycerol gradient sedimentation, and each fraction was separated on an SDS gel, which was stained with Sypro Ruby. Arrows indicate the location of the endogenous Ltp26, Ltp28, and the ectopically expressed Ltp28-TAP bands. The three 55–60-kD associated bands are indicated by an open arrow. (D) Upper panel, the fractions in C were subjected to adenylation and fractionated in a 4%–12% native gel. The location of the adenylated complexes is indicated by an open arrow. Lower panel, RNA was extracted from each fraction, the gRNA labeled with [αP32] GTP in the presence of guanylyltransferase and separated on 12% acrylamide/urea. The gRNA band is indicated. RNA from total mitochondrial extract (Ex) was used as a control.
