GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S3: GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation. Sas6 specifically interacts with both Ana2-WT and Ana2-S38A when the proteins are pre-phosphorylated by Plk4. GST, GST-tagged wild-type (GST-Ana2-WT) or the S38A substitution mutant (GST-Ana2-S38A) were treated with either MBP-Plk4 or MBP-Plk4KD and incubated in vitro with 35S-Met-labeled Sas6 produced in a coupled in vitro transcription/translation reaction. The resulting complex was analyzed by SDS-PAGE and autoradiography

List of site-directed mutagenesis primers from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

List of site-directed mutagenesis primer

Plk4 phosphorylation induces a band-shift in Ana2 from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S1. Plk4 phosphorylation induces a shift in the electrophoretic mobility of Ana2. A. Incubation of Ana2 synthesized by coupled in vitro transcription and translation (IVTT) with active MBP-Plk4 in the presence of increasing ATP induces a distinct band-shift. B. The shift in mobility of Ana2-FLAG protein following its co-overexpression with active non-degradable Plk4 (Plk4ND) is abolished by treating the extract with λ-Phosphatase. C. Alanine substitutions in mass-spectrometry-identified phospho-sites with highest spectral count following in vitro phosphorylation, including those of the STAN motif, still retain the band-shift. D: Phosphorylation sites identified in Ana2 by mass-spectrometry. Serine or threonine residues identified following phosphorylation by MBP-Plk4 in vitro are highlighted in blue. Sites identified as phosphorylated in vivo on tagged-Ana2 (Protein A, FLAG, or GFP tags) purified from D.Mel-2 cells or early Drosophila embryos are highlighted in yellow. The STAN-motif is highlighted in grey. E. The phosphorylation-site responsible for the band shift is located in the N-terminal 280 amino acids of Ana2. Ana21-280, but not Ana2281-420 displays the band-shift in both the co-overexpression assay (upper) and the in vitro phosphorylation assay (lower). F. Band-shift assay using IVTT product of WT and non-phosphorylatable mutants of Ana2 for the sites highlighted in yellow within the N-terminal 280 amino-acid part. The band-shift is seen for each of these mutants

Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S2. Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. Phospho-Mascot-interpreted fragmentation spectra of the peptide 13-LAPRP…EV (phospho-S-38) ILFG…SPR-65 with phosphorylated serine at the position 38 (highlighted red). Labelled peaks correspond to fragment ions. Despite the relatively long peptide, the fragmentation pattern unambiguously identifies the phosphorylation site

Expression levels in Ana2-Myc cell lines from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S4: Expression levels in Ana2-Myc cell lines: An immuno-blot showing comparable levels of expression between an Ana2-WT-Myc and an Ana2-S38A-Myc cell line. The same three cell lines are presented as in Fig. 6B: Untransfected D.Mel-2 cells (lane 1), pAct5-Ana2-WT-Myc (lane 2) and pAct5-Ana2-S38A-Myc (lane 3). Top panel: anti-Asl immuno-blot as a loading control. Bottom panel: anti-Ana2 immuno-blot, revealing endogenous Ana2 and overexpressed Ana2-Myc