FIG. 1.

Isolation of the six-protein Holo-Elongator complex. (A) TAP of Elongator from strains containing either no tagged protein or TAP-tagged versions of Elp1, Elp2, and Elp3 in buffers containing 125 mM NaCl was performed. The purified preparations were then analyzed by SDS-PAGE and silver staining. In each case, the tagged proteins copurified stoichiometrically with five additional proteins, including three uncharacterized polypeptides with apparent molecular masses of 50, 38, and 31 kDa. We have named the new subunits Elp4, Elp5, and Elp6. Coomassie blue staining also revealed that all six proteins were seemingly stoichiometric (data not shown). The three different electrophoretic forms of Elp1 may be a consequence of degradation or posttranslational modifications. Each purified polypeptide was identified by trypsin digestion and MALDI-TOF mass spectrometry. (B) Purification of Holo-Elongator using TAP-tagged Elp4 and Elp5. The presence of the additional subunits in Elongator was confirmed by TAP tapping the fourth and fifth largest components of the complex. Following purification in buffers containing 125 mM NaCl, all six subunits were isolated in each case.