In oocytes of the newt Pleurodeles waltl, the maternal nuclear protein PwA33 occurs on the lampbrush chromosomes and in some nucleoplasmic particles of the germinal vesicle. PwA33 is a modular protein and we used site-directed mutagenesis to alter the sequences encoding two metal-binding regions, the C3HC4 (or RING finger) and B-box motifs. Several mutant clones were generated and their synthetic transcripts were injected into Pleurodeles oocytes for in vive analysis. In the oocyte, all translation products localized in the germinal vesicle. Proteins encoded by RING finger mutant clones were distributed in a pattern identical to that of the wild type protein, but when His266 of the B-box was mutated. PwA33 failed to localize in the lampbrush chromosomes and the nucleoplasmic particles. Using an in vitro colorimetric assay, we demonstrated that PwA33 is a zinc-binding protein and that mutations in the RING finger and B-Box altered its metal- binding properties. The RING finger motif bound two Zn2+ ions and the binding ratios of several mutants were consistent with the tertiary structure recently proposed for this motif. The B-box coordinated one Zn2+ and this binding was inhibited by the His266 mutation. The failure of the His266 mutation to bind zinc and to localize properly within the germinal vesicle suggests that an intact B-box is required for normal functioning of the PwA33 protein in the oocyte.
ASJC Scopus subject areas
- Cell Biology