The composition of the nucleoplasm determines the behavior of key processes such as transcription. However, we only recently were able to quantify the proteome's nucleocytoplasmic partitioning on a genome-wide scale, using the large frog oocyte (Wühr et al. 2015). This oocyte allows rapid manual nuclear isolation without gain or loss of material (see nuclear isolation movie: nucleus manual isolation). While it is an important step forward that we finally have a comprehensive picture of nucleocytoplasmic partitioning in the frog oocyte, we still don't know how much nuclear composition and nuclear localization mechanisms differ in different cell types and species. Furthermore, while we know of several examples where the subcellular localization of an individual protein provides key cues in development, stress response, and general signaling, we now have the ability examine the the consequences of differences in subcellular partitioning of the proteome (~9,000 proteins) on biological function.
How are different compositions of nucleus and cytoplasm maintained in tissue culture cells?
Taking advantage of the quick and reliable nuclear isolation in the frog oocyte, we were able to show that the maintenance of nucleocytoplasmic partitioning in the oocyte is dominated by the assembly of proteins into larger complexes. We combined the measurement of subcellular localization with the ability to measure native molecular weight on a proteome-wide scale. Native size is the size of a protein inside the cell: the size of itself plus any other protein(s) that bind to it. When we integrated these data we found that essentially all proteins that are residing exclusively in the nucleus or the cytoplasm have a native size larger than ~100kDa. This suggested that the main mechanism by which a cell maintains different nuclear and cytoplasmic composition is by complex assembly, which prevents diffusion of proteins through the nuclear pores. We confirmed this finding by inhibiting the canonical nuclear exportin and found that only ~3% of the proteome changed localization in response to this perturbation. While these findings are interesting in their own right, the large oocyte is a highly specialized cell and we don't know if these findings extend to other smaller cell types. Currently, we are interested in making the same measurements in much smaller mammalian tissue culture cells. Our recent development of a quick and reliable nuclear isolation protocol in these cells brings this question within technical reach.
How is the nuclear proteome affected by size-scaling and developmental progression?
Upon fertilization, the nucleus is tiny compared to total cellular volume (see figure below). The nucleocytoplasmic volume ratio (N/C-ratio) only reaches its usual level after many rounds of cell division. We recently developed a reliable nuclear isolation method for small embryonic nuclei. We will combine this technique with the quantitative proteomics methods previously applied to the oocyte. The resulting data will allow us to answer questions like: How does nuclear composition change with different N/C-ratios? Are proteins with high affinity for importins enriched in the early small nuclei? How similar are the nuclei of different types of tissue culture cells? What allows the oocyte to reprogram somatic nuclei to toti-potency?
Predict nucleocytoplasmic partitioning on proteome-wide scale
To understand how the proteome partitions between nucleus and cytoplasm we have to be able to predict the flux over the nuclear envelope, which consists of passive transport (i.e. diffusion) and active transport ( i.e. shuttling via importins and exportins). Recent advances of quantitative proteomics allow us to make these measurements on a proteome-wide scale. We cannot only identify and quantify each individual protein species but also thousands of sites where they are post-translational modified by e.g. phosphorylation or ubiquitination.
To date, the permeability of the nuclear envelope has only been addressed for a few proteins and dextrans. To obtain a comprehensive understanding, we plan to characterize the nuclear-pore permeability for hundreds of prokaryotic proteins, via multiplexed proteomics. Prokaryotic proteins are presumed to be inert to the nuclear transport machinery. Accurate measurements of nuclear envelope flux for many prokaryotic proteins will enable us to systematically determine how nuclear equilibration kinetics relate to native protein size and other physiochemical properties.
To elucidate the role of active nuclear transport, we plan to identify cargoes for all importins and exportins (karyopherins), and measure their binding affinities. We intend to integrate binding affinities with our subcellular localization measurements and try to predict each protein’s subcellular localization from the strength of the karyopherin(s) interaction, karyopherin expression levels and also attempt to predict binding affinities for cargoes from their amino acid sequence.