A vector for double epitope tagging with a recyclable marker.

Multimeric protein complexes play diverse and vital roles in the cell, but following the composition of these complexes under varying growth conditions can be challenging. Toward that goal, we have designed a vector that permits the double epitope tagging of a protein at its carboxy terminus. One 'universal' tag, a triple repeat of the HA1 epitope, is fused with every protein to be studied, allowing the composition and stoichiometry of the proteins in a complex to be detected with a single antibody. Each protein also can be tagged with a second epitope specific for that protein. This 'specific' tag can be used to immunoprecipitate complexes containing that protein of interest. Any epitope to which a specific antibody is available can be used for this second tag. Because there are a limited number of selection markers for cloning in yeast, the kanamycin cassette, flanked by loxP sites, was incorporated into the vector to permit marker recycling using Cre-lox recombinase. This vector was used to tag 4 proteins involved in ribosome biogenesis-Ytm1, Cic1, Brx1 and Drs1. An anti-HA1 antibody could detect all four proteins in crude lysates and yielded the relative abundance of these four proteins, of which Drs1 is reproducibly less abundant than any of the others, which may have implications for the control of ribosome biogenesis. The Ytm1 protein was also tagged with the VSV epitope and can be specifically detected using an anti-VSV antibody. This vector may prove useful for exploring other protein complexes.