Year-end Sale % OFF 
Abstract
The nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, approximately 15-20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170(979-1502) and hNup107(658-925) x hNup133(517-1156), each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and Nup133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major alpha-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication.
Highlights
The architecture of the nuclear pore complex (NPC) is roughly conserved among eukaryotes, measuring ϳ100 nm in the outer diameter, with a central transport gate ϳ40 nm wide [5,6,7,8]
The structural similarity between these ACE1 proteins provided the proof that the NPC and COPII coat derive from a common ancestor [32], as hypothesized previously [42, 43]
Because the NPC is assembled from subcomplexes in a modular fashion, the high resolution structure can be approached by a divide-and-conquer strategy [4]
Summary
Protein Expression and Purification—Nup170 from Saccharomyces cerevisiae was cloned into a pET-Duet vector (Novagen) encoding an N-terminal, human rhinovirus 3C (HR3C)cleavable His tag. YNup1701253–1502 crystallized at 90 mg mlϪ1 in 1-l hanging drops over 0.1 M Tris-HCl, pH 8.5, 0.2 M Li2SO4, 50 mM NaCl, 22–24% PEG 3,350 within 3–5 days at 18 °C. The crystals were grown in drops of 1 l of protein, supplemented with 2% PEG 3,3350, ϩ 1 l of reservoir of 0.8 –1.0 M sodium/potassium phosphate, pH 7.8, 15% glycerol at 18 °C, streak-seeded after 12 h with microcrystals. To improve model quality and attempt to refine against the twinned data diffracting to higher resolution, the C-terminal subdomain yNup1701253–1502 was crystallized, and 2.2 Å data was collected at Beamline 24-IDC at the Advanced. A molecular replacement solution for yNup1701253–1502 was found using a partial model from the initially obtained 3.2 Å structure of yNup170979 –1502.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
