Figure 4.

Upon removal of phosphate modifications acquired during egg arrest at metaphase, soluble ZC3HC1 can re-engage in specific physical interactions with TPR even in solution. (A) IP of TPR and ZC3HC1 from 250,000× g supernatants of X. laevis egg extracts competent for post-mitotic nuclear assembly. IP of NUP62 from the same supernatant was performed as a control in parallel. Lanes were loaded with the total soluble cell proteins (T), with those proteins that had remained unbound (U) after bead incubation, those released during the third of three successive washing steps (W), and those obtained after final elution (E). Loadings in T and U represented the same volume fraction of the respective samples’ total amount (1 V), while the loadings in lanes W and E represented three-fold higher relative amounts (3 V). Immunolabelling was on the upper and lower parts of the PS-stained membrane shown here and on an identical duplicate. Cases in which a protein had been co-immunoprecipitated with the IP’s actual target protein are framed with brackets in green; brackets in magenta accentuate those in which no co-IP had occurred. Note that a subpopulation of TPR polypeptides had been co-immunoprecipitated together with ZC3HC1 and that, vice versa, a large proportion of the extract’s total content of ZC3HC1 had been co-immunoprecipitated with TPR. By contrast, neither TPR nor ZC3HC1 had been isolated together with NUP62, or vice versa. As an aside, note that some ZC3HC1 was found gradually detaching from the immunoprecipitated TPR during washes (blue arrowhead). Such polypeptides appear to represent one of seemingly two ZC3HC1 populations attracted by TPR, of which one is less tightly associated with TPR in vitro than the other. (B) IB of ZC3HC1 as the protein co-immunoprecipitated with TPR, as a component of manually isolated NEs from Xenopus stage V oocytes, and as a protein within the extract from metaphase-arrested Xenopus eggs, with such extract having been left untreated (t0), and after incubation for 15 min (t15) with λ phosphatase alone, and with the same amount of λ phosphatase supplemented with phosphatase inhibitor. The corresponding PS-stained membrane lacked the proteins below 30 kDa due to intentionally prolonged SDS-PAGE for better separation of ZC3HC1 polypeptides. Note that ZC3HC1 dephosphorylated with λ phosphatase exhibited electrophoretic mobility similar to that of the NB-associated ZC3HC1 polypeptides and those co-immunoprecipitated together with TPR from assembly-competent egg extracts.