FIG. 1.

Characterization of the TAP complexes. (A) Protein lysates from the Cdc5p-TAP-producing strain (upper panel) or the Cdc5p-TAP complex following its purification (lower panel) were resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction 1). These were resolved by SDS-PAGE and immunoblotted with anti-Cdc5p serum. The migrations of FAS (40S), thyroglobulin (19S), and catalase (11.3S) collected from parallel gradients are indicated. (B) Silver-stained gel of fractions of the 10 to 30% sucrose gradient resolving the Cdc5p-TAP complex. Asterisks indicate fractions containing multiple peptides. Brackets labeled with the letter K indicate trace amounts of unavoidable human keratin contamination. (C) Silver-stained gels of a portion of each TAP complex. The protein compositions of mock purifications from wild-type S. pombe (KGY246) and wild-type S. cerevisiae (YPH09) were also examined by silver staining. (D) Electron microscopic analysis of the Cdc5p-TAP complex negatively stained with 0.75% uranyl formate. Shown is a gallery of selected particles representing different views of the complex. (E) Cdc5p-TAP associates with U2, U5, and U6 snRNAs. RNA was isolated from Cdc5p-TAP and from an anti-snRNA cap (antitrimethylguanosine [m₃G]) immunoprecipitation from wild-type cells. Blots were probed with ³²P-labeled oligonucleotides complementary to the S. pombe U1, U2, U4, U5, and U6 snRNAs. In the cases of the U2, U5, and U6 snRNAs for the TAP samples, exposures were 1/24 of the others. (F) Prp19p-TAP associates with U2, U5, and U6 snRNAs. RNA was isolated from Prp19p-TAP and from an anti-snRNA cap (anti-m3G) immunoprecipitation from wild-type cells. Blots were probed with ³²P-labeled oligonucleotides complementary to the S. cerevisiae U1, U2, U4, U5, and U6 snRNAs. In the cases of the U2, U5, and U6 snRNAs for the TAP samples, exposures were 1/12 of the others.