Mak11 and Rlp24 are functionally and physically linked. (A) RLP24 is a high-copy-number suppressor of the mak11-2 ts phenotype. Rlp24 was expressed from a high-copy-number vector (pCM190-RLP24) under the control of a tetracycline-repressible promoter in wild-type or mak11-2 cells at permissive and nonpermissive temperatures. Growth was estimated by 10-fold serial dilutions on solid yeast extract-peptone-dextrose medium with or without doxycycline. (B) Protein-protein interaction between (His)6-Mak11 and GST-Rlp24 was detected by mixing total extracts of E. coli overexpressing the proteins and pulling down the GST fusion proteins with glutathione-Sepharose. Eluates were separated on 4 to 12% Novex polyacrylamide gels and Coomassie blue stained. Immunoblot assays were performed in parallel with rabbit polyclonal antibodies raised against Mak11. Asterisks indicate GST-tagged proteins. Rpl3 and Rpl5 were used as controls.
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