Sunday, April 28, 2024
4/28/2024
PROJECT SUMMARY/ABSTRACT The eukaryotic cell has evolved to compartmentalize DNA within the nucleus, surrounded by the barrier of the nuclear envelope (NE), providing protection to the DNA and a means to control information flow to and from the genome. This flow, namely the passage of small molecules, RNAs, and proteins across the NE, is mediated by large, 8-fold symmetrical structures known as nuclear pore complexes (NPCs). Arranged as a stack of rings, the NPC scaffold braces open pores within the NE, yet preserves its barrier function with a dense network of disordered protein domains, rich in repeating phenylalanine-glycine motifs (FG repeats), known as the NPC central transporter. The largest NPC cargoes, including pre-ribosomal subunits, mRNPs, and proteasome subcomplexes, approach the width of the ~55 nm NPC channel in size, yet are able to transit the NPC central transporter. That such large cargoes can traverse the NPC suggests that organized mechanisms and alternate structural states of the NPC are required to transport large cargoes through the FG repeat network of the central transporter. To address these questions, our multi-investigator, multi-site collaborative team recently determined structures of the affinity isolated S. cerevisiae budding yeast NPC at 8-11 Å resolution and the in situ yeast NPC at 30-40 Å resolution (Akey et al., 2022). Comparison of “ground state” (isolated) and “active state” (in situ) NPCs revealed that radial dilation of the NPC scaffold may accommodate changes in cargo flux or size. We now aim to investigate the dynamics of NPC transport function of model large cargoes that can be targeted for import into the nucleus and arrested during transport, allowing us to quantify cargo transport dynamics (function) and map the location of the transiting cargo within the NPC (structure). We will dissect NPC transport’s energy- dependence on nuclear Ran-GTP into separate studies of passive, Ran-insensitive cargo targeting to the NPC (Aim 1) and active, Ran-dependent cargo transport through the NPC (Aim 2). Using well-characterized NPC transport mutants, we will examine the behavior of actively transporting large cargoes by perturbing specific stages along the transport path (Aim 3). This level of control will allow us to map transport functions not only to specific Nups, but to specific regions of Nups. Quantitative fluorescence microscopy and cell fitness assays will screen for functionally important phenotypes to selectively pursue NPC-cargo interactomics by mass spectrometry and visualization of NPC structural changes by cryo-electron microscopy. Our functional and structural data will inform each other in a synergistic but non-dependent fashion to reveal how the NPC adopts distinct and discrete structural states to perform its transport functions for native (mRNPs, pre-ribosomes) and non-native (viral capsids, nanocarriers) large cargoes. This comprehensive research strategy will advance our understanding of (i) constitutive large cargo transport processes and malfunction of this process in disease states (ii) interactions between viral capsids and NPCs during infection and (iii) nuclear-targeting for nanocarriers in drug delivery and gene therapy.
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