We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8: [81][82][83][84][85][86][87][88][89][90][91][92][93] 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.Septins are conserved GTP-binding and filament-forming proteins (51). Septins were discovered in Saccharomyces cerevisiae and later found to be ubiquitous in metazoans (39,53). The cellular functions of septins are diverse and include participation in cytokinesis (22, 40), establishment of diffusion barriers for proteins and mRNAs during cell division (16, 75), vesicle trafficking, exocytosis (7,28,33,37), and apoptosis (29,49). Accordingly, cellular localizations of septins are diverse and include the bud neck (14), division furrow (1), presynaptic vesicles (86), dendritic spines (73, 85), and mitochondria (50, 74). Septins interact with microtubules (45, 71) and microfilaments (46, 56) as well as the endoplasmic reticulum (ER) and filaments emanating from the Golgi network (54, 71). Recently, the diverse patterns of septins were classified into types: projections, partitions, and dispersal over whole cells (52). The molecular functions of septins remain unclear (reviewed in reference 81). So far, septins have been studied only for fungi and metazoans. Since septins are known to be involved in cytoskeletal organization and membrane remodeling events, we anticipated that Tetrahymena thermophila septins are involved in partitions of distinct domains in the structurally elaborate cell cortex of ciliates, in particular during cytokinesis, when the cortex undergoes longitudinal segmentation. Surprisingly, we have found that septins of Tetrahymena thermophila localize to mitochondria and regulate mitochondrial dynamics.
MATERIALS AND METHODSBioinformatics. Protein sequences of the known septins were obtained from the following databases: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Nannochloris bacillaris, Strongylocentrotus purpuratus, Danio rerio, and human sequences from NCBI (National Center for Biotechnology Information, http: //www.ncbi.nlm.nih.gov/); Drosophila melanogaster sequences from FlyBase (http: //flybase.bio.indiana.edu/); Caenorhabditis elegans sequences from the Sanger Institute (http://www.sanger.ac.uk/Project...
