Networks of protein interactions at the nuclear pore complex in Saccharomyces cerevisiae

One of the distinguishing biological features that differentiates eukaryotes from prokaryotes is the compartmentalization of cell functions. A primary example of such compartmentalization is the separation of the nucleus from the rest of the cell. Separation is achieved by the nuclear envelope, a double membrane layer, contiguous with the endoplasmic reticulum. The nuclear envelope is perforated by small channels termed nuclear pore complexes (NPCs) that serve as the only known entryway for macromolecular exchange between the nucleus and the cytoplasm. The rapid trafficking of molecules through the NPC is facilitated by soluble transport factors termed karyopherins. The NPC is comprised of nucleoporins and a specific subset, containing FG di-peptide repeats, is thought to act as the docking sites for the karyopherins during the translocation process. The entire transport process would not be complete without the small GTPase Gsp1p and its effectors, which impart directionality to the transport process by modulating the interactions between karyopherins, cargoes and FG-nucleoporins in a compartment-dependent manner.; In an effort to determine the many protein interactions occurring at the nuclear pore complex in Saccharomyces cerevisiae, we turned to biochemical and proteomic techniques. We used bacterially expressed fusions of GST with components of the nucleocytoplamsic machinery, nucleoporins, karyopherins and Gsp1p GTPase system as bait to capture interacting proteins from yeast extracts which were then identified using mass spectrometry. Using FG Nup fusions and yeast extracts, we were able to reconstitute molecular interactions occurring at the NPC in vitro and generate a basic route that different karyopherins take in their translocation across the pore. We show that specific FG nucleoporins are specialized for binding particular karyopherins, while others serve as more generic docking sites. Manipulation of yeast extracts with Gsp1p-GTP to reflect a nucleoplasmic environment yielded global changes in the karyopherins bound and established a governing role of Gsp1p-GTP in nucleoporin-karyopherin interactions. Using purified protein constituents, we resolve nearest neighbor interactions and provide clues to how protein complexes are tethered directly to the pore. From the results presented here we have described and provided a rudimentary network of protein interactions occurring at the NPC in S. cerevisiae.