X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis.

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.

Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.