Michael Rexach - US grants

DESCRIPTION (provided by applicant): Karyopherin-mediated protein transport across the nuclear pore complex (NPC) is vital for eukaryotic cells yet the mechanisms of karyopherin translocation across the NPC are unsolved. Tumor suppressor proteins, hormone receptors, and cell-cycle checkpoint control proteins are among the hundreds of essential regulatory proteins that need access across the NPC before executing their function in the nucleus. Thus, it is important to human health issues that we achieve a better understanding of the general mechanisms of nucleocytoplasmic transport. The long-term goal of this project is to understand at a molecular level how karyopherins use their interaction with nucleoporins to move across the NPC while carrying cargo. The experimental strategy is to use chemical crosslinkers to identify Nups that function as "stepping stones" for Kap movement within the NPC, then to characterize in detail the interaction between karyopherins and identified nucleoporins using biochemical techniques, and finally to use the knowledge gained from biochemical analyses to design and conduct experiments that will test in vivo the mechanics of karyopherin movement within the NPC. The yeast S. cerevisiae will be used as a model eukaryote for this research. The specific aims are: i) to test the hypothesis that nucleoporins containing FG repeats function as sequential "stepping stones" in the movement of Kap95p across the NPC, ii) to test the hypothesis that nucleoporins are specifically arranged within the NPC to display a "gradient of affinities" for Kap95p that promotes its movement across the NPC, iii) to identify point mutations in Kap95p that interfere with its ability to dock at distinct Nups and test their effects in vivo, and iv) to conduct a comparative study (as delineated for Kap95p) for two additional karyopherins (the exportin Crm1 p, and the importin Kapi 04p) with the goal of uncovering general and specific features in the paths of karyopherin movement in similar and opposite directions across the NPC.

ABSTRACT NOT PROVIDED

mass spectrometry

The nuclear pore complex (NPC) is one of the most important molecular machines in eukaryotes because it gates the porous conduits between the cytoplasm and nucleoplasm of cells and controls all nucleo-cytoplasmic traffic and communication. Its most important architectural feature is a poorly understood semi-permeable diffusion barrier in its center that maintains a tight seal against cytoplasmic proteins as small as 4 nanometers in size, but opens to allow facilitated transport of particles of all shapes and sizes up to 40 nanometers in size. This flexible barrier is composed of a family of filamentous proteins named FG nucleoporins (FG nups) that feature large unfolded domains in their native functional state, which are decorated with multiple phenylalanine glycine motifs (FG domains). The specific aims of our proposed research are to 1) characterize the dynamic structure and intra-molecular interactions of FG domains representing two different types of FG nup filaments that are anchored at three different locales of the NPC, and 2) test the hypothesis that inter-molecular associations between FG domains of nups create a filamentous meshwork structure at the NPC center, which establishes the size-selective barrier to the passive diffusion of proteins. The proposed experiments will combine biochemical, biophysical, cell biological, structural (Nuclear Magnetic Resonance), and molecular modeling techniques to gain insight into the dynamic behavior, structure and function of the FG nups. We will also gain fundamental knowledge on the dynamic behavior and structure of disordered domains of proteins in general. We are studying the three-dimensional structure of the cellular proteins that function as gatekeepers of our genetic material in the nucleus. Their proper architecture and function is vital to human health because they control the flow of information to and from our genes. When they fail to function normally, these proteins can trigger the onset of cancer. We wish to understand how their structure enables them to function as gatekeepers of the nucleus.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to examine the three-dimensional shape of ten different natively- unfolded domains of nucleoporins (nups) by SAXS at different temperatures to confirm and extend our findings that some of these unstructured domains adopt compact configurations that swell upon chilling (similar to tropoelastin), while others adopt highly-extended configurations, depending on their amino acid composition.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structural studies of membrane proteins are limited by the availability of crystals diffracting to high resolution. Crystallization in lipidic cubic phase (LCP) matrices (or in meso) has proven to yield high quality crystals of challenging membrane proteins, such as human G protein-coupled receptors. Broader applications of in meso techniques require identification of new lipids with specific phase properties capable of stabilizing proteins with large range of sizes and architectures. At the Joint Center for Innovative Membrane Protein Technologies (JCIMPT) we are working on design and synthesis of such lipids. The phase and structural behavior of novel lipid matrices should be thoroughly characterized prior to being used in specific applications. We propose to overcome the obstacles associated with conventional preparation of lipid samples for x-ray studies, by preparing samples in 96-well sandwich plates and measuring them in situ at the BioSAXS beamline. This approach will allow us to screen for effects of detergents, additive lipids, proteins as well as great variety of precipitants on the lipidic matrices in the high-throughput mode at conditions mimicking those encountered during crystallization trials. During the duration of this proposal we anticipate to fully characterize 10-12 most promising novel lipids pre-selected out of a larger pool of synthesized candidates. The obtained results will be indispensable for selection of proper lipids and guiding in meso crystallization experiments.