Cross-reactivity of antibodies to actin- depolymerizing factor/cofilin family proteins and identification of the major epitope recognized by a mammalian actin-depolymerizing factor/cofilin antibody.

Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica.

A key role for the alpha 1 helix of human RAP74 in the initiation and elongation of RNA chains.

RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the alpha1 helix of the RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript, mutations within the alpha1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74. Changes within the N-terminal domain include disruptions of specific contacts with the alpha1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan, S., and Richmond, T. J. (2000) J. Mol. Biol. 302, 1119-1127). Contacts between the beta4-beta5 loop and the alpha1 helix are shown to be largely unimportant for alpha1 helix function. Other mutations throughout the N-terminal domain are consistent with the establishment of the dimer interface with the RAP30 subunit. The RAP74-RAP30 interface is important for TFIIF function, but no particular RAP74 amino acids within this region have been identified that are required for TFIIF activities. The molecular target of the alpha1 helix remains unknown, but our studies refocus attention on this important functional motif of TFIIF.