Rei1 is physically associated with nontranslating, Rpl24-containing complexes. (A) Rei1 is a two-hybrid prey of Rpl24b. YBR267W (REI1) was found as prey ORF in a genomic two-hybrid screen performed with RPL24B as bait. We represented the 393–amino acid–long protein and pointed out its putative motifs: three conserved C2H2 Zn fingers (dark gray) and the PFAM E-MAP-115 motif (stripes). Underneath, displayed as black rectangles, are the seven distinct DNA portions of REI1 that were found as preys. The minimal interacting domain is indicated in light gray. (B) Rei1-myc copurifies specifically with Rpl24b-TAP. TAPs were performed with strains expressing Rei1-13Myc together with Ssf1-TAP, Rlp24-TAP, Rpl24-TAP, or in a nontagged strain. The components of the purified complexes were separated by SDS-PAGE as shown on the Coomassie staining of the TEV eluate. Rei1-13Myc and Nog1 were detected in the TEV eluates by immunoblotting. As a loading control, Rei1-13Myc was detected in the input fractions (top). (C) Rei1 is associated with 60S particles. Whole cell extracts were prepared from a strain expressing Rpl24b-TAP and separated on a sucrose gradient by ultracentrifugation. Absorbance at 254 nm was measured, and 0.5-ml fractions of the gradient were collected. Peaks corresponding to the 40S, 60S, and 80S particles or to the polysomes are indicated. The proteins of each fraction were TCA precipitated and analyzed by Western blotting with antibodies directed against Rei1 or with PAP for the detection of the TAP fusion protein.
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