ABSTRACT
Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an â¼50 MDa assembly comprised of â¼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.
Subject(s)
Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high-resolution structures of large, multisubunit tethering complexes that spatially and temporally control intracellular membrane fusion. Exocyst is a large hetero-octameric protein complex proposed to tether secretory vesicles at the plasma membrane to provide quality control of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence microscopy, and single-particle cryoelectron microscopy, are providing critical insights into the structure and function of the exocyst. These studies now pose more questions than answers for understanding fundamental functional mechanisms, and they open wide the door for future studies to elucidate interactions with protein and membrane partners, potential conformational changes, and molecular insights into tethering reactions.