The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.
Bottom Line: We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1.SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes.These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.
Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.Show MeSH
Mentions: To identify potential cytoplasmic interactions of the PCM proteins SPD-2 and SPD-5, we used label-free-quantitative mass spectrometry. For proper quantification of protein amounts using this technique, the immunoprecipitated proteins must carry the same tag. We therefore used green fluorescent protein (GFP)–tagged versions of SPD-2 and SPD-5 (GFP::SPD-2 and SPD-5::GFP, respectively), which rescue depletion of the corresponding endogenous proteins and therefore are functional (Figure 1, A and B). First, we used ultracentrifugation to generate centrosome-free cytoplasmic extracts from transgenic C. elegans embryos expressing GFP::SPD-2 and SPD-5::GFP, similar to what was described by Gopalakrishnan et al. (2011). GFP::SPD-2 or SPD-5::GFP was then immunoprecipitated from these extracts using bead-coupled GFP antibodies and eluted, and then the coprecipitating proteins were identified by mass spectrometry; each experiment was repeated three times. The enrichment and significance of the enrichment of an identified interactor were assessed via label-free quantification using control protein (TBG-1::GFP) immunoprecipitates as described by Hubner et al. (2010). Analysis of SPD-5::GFP immunoprecipitates revealed that, in addition to SPD-5 (2022-fold enriched in comparison to the control), the most enriched proteins were RSA-2 (63-fold enriched) and RSA-1 (29-fold enriched) (Figure 1C), two known regulatory subunits of the protein phosphatase 2A (PP2A) complex (Schlaitz et al., 2007). This result is consistent with previous LAP::RSA-1 immunoprecipitations from centrosome-containing whole-worm extracts by Schlaitz et al. (2007). A novel interaction was found between SPD-5 and the RNA-binding protein RNP-6 (25-fold enriched above simulated noise level; see Materials and Methods), which shows a postembryonic lethal phenotype upon depletion (Sönnichsen et al., 2005). However, since RNP-6 depletion did not affect centrosomes during the first cell division (unpublished data), its role was not investigated further. Of importance, SPD-2, the centrosome-related kinases PLK-1 and AIR-1, and the centriole proteins SAS-4/-5/-6 were not detected in the immunoprecipitates. This data suggest that SPD-5 molecules interact with RSA-1, RSA-2, and RNP-6 in cytoplasmic embryo extracts.
Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.