In fact, we observed this association whether the flagellum is linked to a single kDNA network (Figure 1G) or, if derived from a cell that has finished kDNA replication and initiated segregation, to two networks (not shown). biology, with many characteristics differing from those in more familiar eukaryotes. One amazing feature is usually its unusual mitochondrial DNA, known as kinetoplast DNA (kDNA) (for reviews, observe Shlomai, 2004; Liu of 5.2. Homologous genes are present in related and species but not in other species. Based on software at http://elm.eu.org, p166 has a predicted N-terminal mitochondrial targeting transmission and a predicted transmembrane domain name (residues 1440C1462). Therefore, the protein’s N-terminal region (including the targeting transmission) is usually 1439 residues, its predicted transmembrane sequence is usually 23 residues, and its C-terminal region is usually 39 residues. p166 localizes with the TAC Physique 1A shows three constructs encoding p166 fusion proteins, each with three tandem myc epitopes (comprising a total of 42 amino-acid residues), at a different location at or near the C-terminus. In these studies, we assumed that residues 1440C1462 constitute a transmembrane domain name. Using immunofluorescence (IF) with anti-myc antibody, we found that full-length p166myc (green) localizes between the kDNA (blue) and the flagellar basal body (reddish) (Physique GSK2126458 (Omipalisib) 1B, with enlargement in Physique 1E, panel a; the enlarged image showed a dividing kinetoplast associated with basal body that had already separated). There was a similar pattern in virtually all cells examined from an asynchronous culture (including those with two kinetoplasts), indicating that this localization is independent of the cell cycle. Open in a separate window Physique 1 Localization of p166. (A) Diagrams, not drawn to level, of myc-tagged proteins expressed in genomic locus and used in localization. p166myc is the full-length protein and Cp166myc and TMCp166myc are truncated as indicated. MTS, mitochondrial targeting transmission; TM, predicted transmembrane domain name. 3 GSK2126458 (Omipalisib) myc, three copies of myc epitope. (BCD) Localization of p166myc, Cp166myc, and TMCp166myc, respectively. Fluorescence micrographs used anti-myc antibody to recognize p166 (green), anti-tyrosinated tubulin antibody for basal body (reddish), and DAPI to stain kDNA and nucleus (blue). Boxes are regions enlarged in (E). Level bar, 2 m. (E) Enlargements showing GSK2126458 (Omipalisib) association of kDNA, p166, and basal body. a, p166myc; b, Cp166myc; c, TMCp166myc. (F) Localization of p166myc in isolated cytoskeleton by fluorescence microscopy. Anti-myc antibody detected p166myc (green) and DAPI stained the kDNA and the nucleus (blue). (G) Isolated flagellum stained as in panel F, except that this basal body was also detected by anti-tyrosinated tubulin (TyrCTu). Level bar in panels F and G, 5 m. Fluorescence images were altered uniformly for contrast and brightness with Adobe Photoshop. We GSK2126458 (Omipalisib) next examined Cp166myc (lacking the 39 residue C-terminal tail) and found that its localization closely resembled that of the full-length GSK2126458 (Omipalisib) protein (Physique 1C, with enlargement in Physique 1E, panel b). Surprisingly, TMCp166myc, lacking the putative transmembrane domain name as well as the C-terminal tail (totaling 62 residues), did not redistribute in the mitochondria. Instead, as shown in Physique 1D (with enlargement in Physique 1E, panel c), it experienced a localization much like those in Figures 1B and C. This latter observation raised the possibility that the protein not only traverses the mitochondrial membrane but also is held in place by some other interaction, possibly with a cytoskeletal structure. To assess whether the myc tag might impact p166’s function and/or localization, we myc tagged the products of Plxna1 both alleles. After using PCR to show that each of the two wild-type alleles had been replaced by a sequence encoding p166myc (Supplementary Physique S1, Panel A), we then demonstrated that this myc-tagged protein was actually expressed (see western blot in Supplementary Physique S1, Panel B) and that the double myc tag had no effect on growth (Supplementary Physique S1, Panel C). We also detected no switch in the kDNA, as visualized by DAPI staining (data not shown). Finally, we exhibited that p166myc, in cells generating only the tagged version of the protein, also localizes between the kDNA disk and the basal body (Supplementary Physique S1, Panel D). These data provide strong evidence that this myc tag does not interfere with this p166’s function or localization. p166 stably associates with the TAC The TAC system remains intact following cell lysis with NP-40 (Robinson and Gull, 1991). Therefore, we used IF to examine cytoskeletons (Physique 1F) from cells extracted with NP-40 and MgCl2 (a treatment that removes membranes but does not disrupt.