FIG. 11.

Npa2p forms a discrete complex of about 600 kDa. Strains IVY317 (Npa2p-TAP), IVY325 (Npa1p-HA), IVY347 (HA-Dbp6p), IVY411 (Nop8p-HA), and IVY409 (Rsa3p-HA) harboring a NPA2-TAP construct were grown at 30°C to mid-log phase. (A) Cells were broken in a mortar grinder, processed as ultracentrifuged extracts as described in Materials and Methods, and treated with 100 ng/ml RNase A for 1 h at 4°C. Extracts were then subjected to gel filtration chromatography through a Superose 6 HR column. Fractions were collected and subjected to SDS-PAGE and Western blotting using specific antibodies to detect the different proteins analyzed. T, total loaded extract; numbers correspond to fraction numbers. Arrows indicate elution positions of molecular mass standards. (B) Gel filtration analysis of ultracentrifuged extracts not treated with RNase.