(a) Centrosomes purified from wild-type embryonic extracts contain Sas-4, CNN, Asl, CP-190, tubulin and Sas-6, while stripped centrosomes contain only Sas-6 and tubulin. Purified Sas-4 complexes contain the Sas-4, CNN, Asl, CP-190 and tubulin. (b) Binding assays of purified Sas-4 complexes, Sas-4-N190 or Sas-4-Δ90 to stripped centrosomes and analyses by discontinuous sucrose gradient. Low-density (LD) fractions contain unbound proteins and high-density (HD) fractions contain stripped centrosomes and the proteins bound to them. In the absence of stripped centrosomes, Sas-4 complexes, Sas-4-N190 or Sas-4-Δ90 remain only in low-density fractions. When mixed with stripped centrosomes, Sas-4 complexes and Sas-4-Δ90 but not Sas-4-N190 are found in the HD fractions. Excess complexes and proteins remain in the unbound fractions. (c) Binding assays of Sas-4-Δ90, CNN or a combination of Sas-4-Δ90 and CNN to stripped centrosomes. LD fractions contain unbound proteins, whereas HD fractions contain stripped centrosomes and proteins bound to them. CNN or Asl alone cannot bind stripped centrosomes. In the presence of Sas-4-Δ90 and stripped centrosomes, CNN and Asl are bound to stripped centrosomes. (d) Schematic of the Sas-4 protein domains. Tethering: as Sas-4Δ90 binds to stripped centrosomes while Sas-4-N190 does not, the centrosome-binding domain of Sas-4 appears to be between residues 191 and 901. CNN, Asl, β-tubulin binding depends on the presence of the PN2-3 domain. D-PLP and γ-tubulin binding: as Sas-4-N190 binds to D-PLP and γ-tubulin, these proteins’ binding domain in Sas-4 appears to be between residues 1 and 190. Green box: conserved segment in PN2-3 domain. CP-190 binding: as Sas-4-N190 and Sas-4Δ150 bind to CP-190, the CP-190 domain of Sas-4 appears to be between residues 151 and 190. (e) A model for the role of Sas-4 in PCM formation. Centrosome biogenesis begins with the formation of a nascent procentriole and the assembly of centriole microtubules. S-CAP complexes form in the cytoplasm and are then tethered to a procentriole via Sas-4. The centriole elongates and matures to a functional centrosome. Sas-4 (blue); microtubules (green); PCM (grey); appendages (orange).