Wilhelm bernhard workshop on the cell nucleus

IDENTIFICATION OF PROTEINS INTER-ACTING WITH THE KARYOSKELETAL PROTEIN NO145

Voltmer S., Kneissel S. and Schmidt-Zachmann M.S.

German Cancer Research Center, Heidelberg, Germany
The nucleolus is a large and complex nuclear substructure with a well defined functional organization. Whereas the major function of this membraneless organelle, the ribosome biosynthesis, has been extensively investigated, little is known about the molecular components contributing to the shape and internal architecture of nucleoli.

Recently, we succeeded in the identification of a novel type of karyoskeletal protein and molecular marker for a specific nucleolar substructure, i.e. a relatively thin cortical layer forming a cage-like perinucleolar structure and consisting of a meshwork of patches and filaments. Whereas this protein- termed NO145 - can be classified as a karyoskeletal protein (resistant to high-salt/detergent extraction) it is also sensitive to regulated proteolysis. Remarkably, protein NO145 (originally identified in Xenopus laevis oocytes) is present throughout all stages of oogenesis but is rapidly degraded during meiotic maturation, i.e. egg formation (Kneissel et al. 2001, Mol. Biol. Cell 12, 3904-3918). The major aim of our current studies is the identification of proteins interacting with protein NO145, e.g. by co-immunoprecipitation experiments and yeast two-hybrid screens. We started the project by constructing a Gal 4 AD fusion library using cDNA prepared from X. laevis ovary. The two hybrid screen was performed by co-transformation of this library with full-length protein NO145 used as a bait. We obtained 60 transformants expressing interacting proteins and started to verify these putative interactions. To identify the genes responsible for a positive two-hybrid interaction the corresponding plasmids were isolated, amplified by PCR and finally sequenced. Detailed data base searches resulted in the identification of several known proteins, but also of X. laevis ESTs coding for unknown proteins. Further verifications of these putative interaction partners of NO145 in vitro and in vivo are in progress.