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The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation.

Rogala KB, Dynes NJ, Hatzopoulos GN, Yan J, Pong SK, Robinson CV, Deane CM, Gönczy P, Vakonakis I - Elife (2015)

Bottom Line: Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo.Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain.Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

No MeSH data available.


The SAS-5 N- and C-terminal segments are unstructured in isolation.(A) Overlaid CD spectra of SAS-5 N-terminal (residues 2–122) and C-terminal (residues 269–404) fragments recorded at 10°C. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar chart left. Grey colour corresponds to random coil, green to β-strand and red to α-helical segments. (B) Thermal unfolding profiles of the same samples based on their CD signal at 222 nm.DOI:http://dx.doi.org/10.7554/eLife.07410.008
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fig1s5: The SAS-5 N- and C-terminal segments are unstructured in isolation.(A) Overlaid CD spectra of SAS-5 N-terminal (residues 2–122) and C-terminal (residues 269–404) fragments recorded at 10°C. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar chart left. Grey colour corresponds to random coil, green to β-strand and red to α-helical segments. (B) Thermal unfolding profiles of the same samples based on their CD signal at 222 nm.DOI:http://dx.doi.org/10.7554/eLife.07410.008

Mentions: Interestingly, thermal unfolding of the soluble SAS-5Δ282–295 and SAS-5FLEX variants showed a two-step cooperative melting process, thus revealing the presence of two independently folded domains (Figure 1C). Sequence-based prediction suggested the presence of a coiled coil spanning residues 125–180, as well as of three tightly-spaced α-helices (residues 210–265); these two elements are separated from each other by a presumed disordered linker of ∼30 residues (Figure 1—figure supplement 4). We expressed both the coiled coil (SAS-5CC) and the predicted helical region (SAS-5Imp for Implico, see below) and performed CD analysis. SAS-5CC displayed a double-minimum spectrum (Figure 1D) characteristic of α-helical coiled coils, and exhibited moderate thermal stability in isolation (apparent melting transition temperature, Tm ∼ 37°C, Figure 1E). In contrast, CD of SAS-5Imp revealed a very stable (Tm ∼ 72°C) α-helical domain (Figure 1D,E). A SAS-5 construct that combined both the coiled coil and the α-helical domain (SAS-5125–265, residues 125–265) showed CD spectra and a two-step thermal unfolding profile highly similar to that of SAS-5Δ282–295 and SAS-5FLEX (compare Figure 1F,G with Figure 1B,C). We attribute the first melting transition of SAS-5125–265 (Tm of ∼50°C) to unfolding of the coiled-coil domain and the second (Tm of ∼70°C) to unfolding of the Implico domain. Moreover, CD spectra of isolated SAS-5 N-terminal (residues 2–122) or C-terminal (269–404) fragments showed no persistent secondary structure or cooperative thermal unfolding (Figure 1—figure supplement 5), consistent with similar previous analysis of the SAS-5 N-terminus (Shimanovskaya et al., 2013). Overall, these findings led us to conclude that the segment between residues 125–265 contains two independently folded domains and encompasses all structured elements of SAS-5.


The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation.

Rogala KB, Dynes NJ, Hatzopoulos GN, Yan J, Pong SK, Robinson CV, Deane CM, Gönczy P, Vakonakis I - Elife (2015)

The SAS-5 N- and C-terminal segments are unstructured in isolation.(A) Overlaid CD spectra of SAS-5 N-terminal (residues 2–122) and C-terminal (residues 269–404) fragments recorded at 10°C. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar chart left. Grey colour corresponds to random coil, green to β-strand and red to α-helical segments. (B) Thermal unfolding profiles of the same samples based on their CD signal at 222 nm.DOI:http://dx.doi.org/10.7554/eLife.07410.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471805&req=5

fig1s5: The SAS-5 N- and C-terminal segments are unstructured in isolation.(A) Overlaid CD spectra of SAS-5 N-terminal (residues 2–122) and C-terminal (residues 269–404) fragments recorded at 10°C. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar chart left. Grey colour corresponds to random coil, green to β-strand and red to α-helical segments. (B) Thermal unfolding profiles of the same samples based on their CD signal at 222 nm.DOI:http://dx.doi.org/10.7554/eLife.07410.008
Mentions: Interestingly, thermal unfolding of the soluble SAS-5Δ282–295 and SAS-5FLEX variants showed a two-step cooperative melting process, thus revealing the presence of two independently folded domains (Figure 1C). Sequence-based prediction suggested the presence of a coiled coil spanning residues 125–180, as well as of three tightly-spaced α-helices (residues 210–265); these two elements are separated from each other by a presumed disordered linker of ∼30 residues (Figure 1—figure supplement 4). We expressed both the coiled coil (SAS-5CC) and the predicted helical region (SAS-5Imp for Implico, see below) and performed CD analysis. SAS-5CC displayed a double-minimum spectrum (Figure 1D) characteristic of α-helical coiled coils, and exhibited moderate thermal stability in isolation (apparent melting transition temperature, Tm ∼ 37°C, Figure 1E). In contrast, CD of SAS-5Imp revealed a very stable (Tm ∼ 72°C) α-helical domain (Figure 1D,E). A SAS-5 construct that combined both the coiled coil and the α-helical domain (SAS-5125–265, residues 125–265) showed CD spectra and a two-step thermal unfolding profile highly similar to that of SAS-5Δ282–295 and SAS-5FLEX (compare Figure 1F,G with Figure 1B,C). We attribute the first melting transition of SAS-5125–265 (Tm of ∼50°C) to unfolding of the coiled-coil domain and the second (Tm of ∼70°C) to unfolding of the Implico domain. Moreover, CD spectra of isolated SAS-5 N-terminal (residues 2–122) or C-terminal (269–404) fragments showed no persistent secondary structure or cooperative thermal unfolding (Figure 1—figure supplement 5), consistent with similar previous analysis of the SAS-5 N-terminus (Shimanovskaya et al., 2013). Overall, these findings led us to conclude that the segment between residues 125–265 contains two independently folded domains and encompasses all structured elements of SAS-5.

Bottom Line: Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo.Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain.Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

No MeSH data available.