Spector, A. attributes. A classical example occurs in promoters, where different arrangements of sequence elements result in the recruitment of different combinations of transcription factors that can provide the complex regulation needed for processes such as differentiation and development. Another example is in the repeated use of various polypeptides in different protein complexes. In some cases, such as the TBP-associated factors (TAFs) present in both the transcription factor IID (TFIID) CCT241533 hydrochloride and Spt-Ada-Gen5 acetyltransferase (SAGA) complexes (1, 2), the resulting complexes are involved in the same general process, in this example transcription. In other cases, however, the same proteins can exert their effect in completely different processes; for example, glyceraldehyde-3-phosphate dehydrogenase functions as a glycolytic enzyme in the cytoplasm as well as a member of a nuclear co-activator complex involved in cell cycle-regulated transcription from the promoter (3). This last theme is becoming more and more common as we learn more about the players in various cellular processes. The snRNA-activating protein complex SNAPc is a multisubunit complex containing five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19, that is required for RNA polymerase II and III transcription of the human snRNA2 genes (for a review see Ref. 4). The arrangement of the subunits within the complex has been deduced from protein-protein interaction studies and reconstitution of partial complexes transcription of RNA polymerase II and III snRNA genes, albeit with lower efficiency than complete SNAPc (7). We find that SNAP45, but not the backbone SNAPc subunit SNAP190, localizes to the centrosomes during specific stages of mitosis as well as to the spindle midzone during anaphase and the mid-body during telophase. Both down-regulation and overexpression of SNAP45 result in abnormalities in mitotic progression, Ptgs1 strongly suggesting that besides its role within the transcription factor SNAPc, SNAP45 performs a second essential function during cell division. Thus, SNAP45 is an example of a protein with two very different functions, the first as a subunit of the transcription factor SNAPc (9) and the second as a protein involved in mitosis. EXPERIMENTAL PROCEDURES phosphorylation assays, 5-10 pmol of SNAP45 and, as a positive control, Orc2 (11) were incubated in 40 l of kinase buffer (50 mm HEPES (pH 7.0), 10 mm MgCl2, 4 mm MnCl2, 1 mm dithiothreitol, 0.1 mg/ml BSA where indicated, and CCT241533 hydrochloride 2 Ci of [-32P]ATP) for 30 min at 30 C in the presence CCT241533 hydrochloride of the indicated amounts of either purified cyclin A/Cdk2, cyclin E/Cdk2, or CCT241533 hydrochloride cyclin B/Cdk1 (Upstate). The reactions were stopped with Laemmli buffer and subjected to SDS-PAGE, and the gels were autoradiographed. RESULTS and reflects warping of the gel), and the same was true for SNAP50. Open in a separate window FIGURE 1. Localization of SNAP45 during the cell cycle. reactivity of the anti-SNAP45 antibody. Whole cell extract from mock-transfected HeLa cells (asynchronous HeLa S3 cells were subjected to cell sorting, and gated samples were collected for immunoblot analysis. immunoblot analysis of SNAP45 in cell cycle-staged cells. Samples from asynchronous cells or from cells with G0/G1, S, and G2/M DNA contents were subjected to immunoblot analysis with antibodies directed against cyclin B (BD Biosciences), SNAP45 (SZ2809), SNAP50 (CS303), and -tubulin (clone B-5-1-2, Sigma). HeLa cells were fixed with 2% paraformaldehyde and stained for indirect immunofluorescence with the anti-SNAP45 (SZ2809) (show a blow-up of the midbody region in down-regulation of SNAP45 by siRNAs. HeLa cells were transfected two times in a 24-h interval with a control siRNA and two different siRNAs against SNAP45 RNA. Samples were collected 24, 48, and 72 h after the second transfection and analyzed by immunoblot with anti-SNAP45, anti-SNAP190 (CS696), anti-SNAP50, and anti–tubulin (as a loading control) antibodies as in Fig. 1phase contrast light microscopy pictures showing an accumulation of rounded mitotic cells after depletion of SNAP45 with the SNAP45 S4 siRNA. FACS analysis of control and SNAP45 S4 siRNA-treated cells. down-regulation of SNAP190 by siRNA. HeLa cells were transfected as in but with control siRNA or siRNA SNAP190-2 directed against SNAP190. Samples were collected 24, 48, and 72 h after the second transfection and analyzed by immunoblot with anti-SNAP45, anti-SNAP190, CCT241533 hydrochloride anti-SNAP50, and anti–tubulin (as a loading control).