As a conserved organelle, the centrosome and its regulated biogenesis is required for cilium formation, accurate cell division, and genome integrity (Nigg and Raff, 2009). Centrosomes consist of a centriole pair surrounded by an amorphous protein network known as pericentriolar material or PCM (Nigg and Raff, 2009). PCM assembly is a tightly regulated step during centrosome biogenesis that determines the size and capability of centrosomes (Conduit et al., 2010; Kirkham et al., 2003; Piehl et al., 2004). During PCM assembly, Sas4, one of the PCM proteins activated by tubulin in GDP bound conformation, provides a scaffold for preassembled cytoplasmic complexes to be tethered to the centrosome. Tubulin directly binds to Sas4; together, they are components of cytoplasmic complexes of centrosomal proteins (Gopalakrishnan et al., 2011; Hung et al., 2004). On the basis of the guanine nucleotide bound state, tubulin can act as a molecular switch in PCM recruitment. While tubulin-GTP prevents Sas4 from forming protein complexes, tubulin-GDP promotes it (Gopalakrishnan et al., 2012).
Since tubulin-GDP, instead of tubulin-GTP, promotes formation of centrosomal protein complex recruited by Sas4, it is interesting to note that in a normal, healthy cell, the proportion of GTP: GDP in cytoplasm is near 100:1 (Gamberucci et al., 1994). Thus, in this study, we hypothesize that in order to guarantee proper PCM assembly, Sas4 specifically binds to tubulin-GDP. In other words, Sas4 could be utilized as a binding probe specifically for tubulin-GDP conformation.
Our research focuses on in vitro experiments that identify Sas4-tubulin interaction and the specificity under different guanine nucleotides. A number of in vitro biochemistry assays were carried out in order to test Sas4-Tubulin interaction in different guanine nucleotide statuses. Co-Immunoprecipitation, Pulldown assay, Native PAGE-Far Western and Far Western Dot-Blot methods were utilized to test our hypothesis. The results are consistent with our hypothesis that Sas4 specifically recognizes tubulin-GDP, and it is further confirmed by the fact that tandem-Sas4, a recombinant protein derived from our newly built plasmid, specifically increased the binding strength to tubulin-GDP by at least 50%.
Our results further confirmed that Sas4N specifically binds tubulin-GDP and function as a core in scaffolding centrosomal protein in the maturation of centrosome. Meanwhile , it also give us a potential tool to utilize Sas4, which specifically binds to tubulin-GDP, to gain more details of insights on the function of free tubulin-GDP as molecule switch instead of a building block in biological process like microtubule dynamic instability