Structural Evidence for Common Ancestry of the Nuclear Pore Complex and Vesicle Coats

Abstract

Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eight-fold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the Nup85•Seh1 structure, a module in the heptameric Nup84 complex. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identify interactions that define the organization of the Nup84 complex based on comparison with vesicle coats and confirmed the sites by mutagenesis. We propose the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice providing docking sites for additional nucleoporins.Exchange of macromolecules across the nuclear envelope is exclusively mediated by NPCs (1-3). Whereas much progress has been made understanding the soluble factors mediating nucleocytoplasmic transport, the structure of the ~40-60 MDa NPC itself is still largely enigmatic. Cryo-electrontomography (cryo-ET) and cryo-electronmicroscopy (cryo-EM) have established the NPC structure at low resolution (4-6). Crystal structures of scaffold NPC components are emerging (7-10), but the resolution gap still precludes fitting into the cryo-ET structure. Overall, the NPC has eight-fold rotational symmetry with an outer diameter of ~100 nm and a core scaffold ring ~30 nm wide. The central FG-repeat containing transport channel measures ~40 nm in diameter, defining the maximum size of substrates (11).The modularity of the NPC assembly suggests a path toward a high-resolution structure (12). Of the ~30 bona fide nucleoporins (Nups) that comprise the NPC, only a core subset is stably attached (13). In S. cerevisiae, this core includes two essential complexes; the heptameric Nup84 complex and the heteromeric Nic96-containing complex (hereafter called the Nic96 complex; unless noted all proteins are from S. cerevisiae). The Nup84 complex is composed of one copy each of Nup84, Nup85, Nup120, Nup133, Nup145C, Sec13 and Seh1. It self-assembles from recombinant proteins in vitro and forms a branched Y-shaped structure (14). Deletion or depletion of individual components of the Nup84 complex leads to severe assembly defects in many organisms (15)(16)(17).

Regarding the presence of membrane coat proteins in bacteria: Confusion? What confusion?

“…In addition, and unlike stated in ref. [7], eukaryotic MCs do not all contain clathrin-like repeats but instead display an impressive range of structural variation on a common framework [9]. It is important to state here that despite the homology between eukaryotic MCs, their sequences and structures have diverged almost beyond the point of recognition.…”

“…It is important to state here that despite the homology between eukaryotic MCs, their sequences and structures have diverged almost beyond the point of recognition. Notwithstanding this divergence, it is now broadly accepted that the eukaryotic MCs have a common origin, a prediction we made four years before the first crystallographic support was obtained [8,9]. A structural comparison of the alpha-helical repeats using one of the most accurate tools available today, COPS (topsearch.services.came.sbg.ac.at, [10]), reveals that the predicted structures of the bacterial MCs do not differ more from the eukaryotic MCs than the eukaryotic MCs do between themselves.…”

Functional Insights from Studies on the Structure of the Nuclear Pore and Coat Protein Complexes

“…In some species, up to three additional proteins (Nup37, Nup43, and ELY5/ELYS) extend the Y-complex. Crystal structures of several 40-to 135-kDa fragments of the 575-kDa conserved Y-complex core are available and account for a combined 90% of the structure (Berke et al 2004;Brohawn et al 2008;Leksa et al 2009;Nagy et al 2009; Whittle and Schwartz 2009). All proteins fold into elongated, stacked a-helical domains or b-propeller domains, or binary combinations thereof.…”

“…The first such class is the group of nucleoporins that share the ancestral coatomer element ACE1 (Brohawn et al 2008). ACE1 is a 65-kDa tripartite module of 28 core a-helices.…”

“…Instead of being closed and seven-bladed as most b-propellers are (Chaudhuri et al 2008;Stirnimann et al 2010), they are open and sixbladed. The Sec13 and Seh1 interaction partners, Nup145C and Nup85, respectively, insert a seventh blade into the propeller to close it in trans, generating a substantial interface area that helps to stabilize the interaction (Brohawn et al 2008;. In COPII, Sec13 interacts very similarly with Sec31 and Sec16, respectively (Fath et al 2007;Whittle and Schwartz 2010).…”