Spatial Organization and Molecular Interactions of the Schizosaccharomyces pombe Ccq1–Tpz1–Poz1 Shelterin Complex

Abstract

The shelterin complex is a macromolecular assembly of proteins that binds to and protects telomeric DNA, which composes the ends of all linear chromosomes. Shelterin proteins prevent chromosome ends from fusing together and from eliciting erroneous induction of DNA damage response pathways. Additionally, shelterin proteins play key roles in regulating the recruitment and activation of telomerase, an enzyme that extends telomeric DNA. In fission yeast, Schizosaccharomyces pombe, interactions between the shelterin proteins Ccq1, Tpz1, and Poz1, are important for regulating telomerase-mediated telomere synthesis, and thus, telomere length homeostasis. Here, we used electron microscopy combined with genetic labeling to define the three-dimensional arrangement of the S. pombe Ccq1-Tpz1-Poz1 (CTP) complex. Cross-linking mass spectrometry was used to identify individual residues that are in proximity to the protein-protein interfaces of the assembled CTP complex. Together, our data provide a first glimpse into the architectural design of the CTP complex and reveals unique interactions that are important in maintaining the S. pombe telomere in a non-extendible state.

Structure of the fission yeast S. pombe telomeric Tpz1-Poz1-Rap1 complex

“…Gel-filtration analysis showed that Poz1, Ccq1, Tpz1, and Pot1 form a stable quaternary complex (Supplementary information, Figure S9). A recent study reported that, in addition to Poz1, Ccq1 can also form a homodimer [47]. Our yeast two-hybrid analysis confirmed that the C-terminal coiled-coil domain of Ccq1 (residues 439-735) indeed mediates homodimerization interaction (Supplementary information, Figure S10).…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…For instance, cross-linking has been successfully coupled with phylogenetic sequence alignment to identify potential evolutionarily-conserved, functionally important residues. Subsequent mutation studies of candidate sites confirmed loss-of-function mutants, permitting localization of protein interaction interfaces in Schizosaccharomyces pombe telomere complexes 97,100 . Similarly, cross-linking data was used to direct truncation/deletion mutagenesis for identification of protein binding domains involved in exocytosis and microtubule complexes 140 .…”

“…Both types of acquisition methods provide comparable and complementary results during data-dependent analyses. This MS n -based workflow has been successfully applied to map protein-protein interactions and elucidate architectures of protein complexes in vitro and in vivo 74,79,95–100 .…”

IL-6, IL-17 and Stat3 are required for auto-inflammatory syndrome development in mouse

“…Stat3 is also reportedly required for arthritis development in RA 37 – 39 . By contrast, inflammasome activation followed by expression of inflammatory cytokines by macrophages 40 reportedly promotes pathogenesis of auto-inflammatory syndromes 41 – 43 . However, inflammasome-independent auto-inflammatory syndromes have also been reported 44 , 45 ; thus not all mechanisms underlying auto-inflammatory syndromes have been defined 46 .…”