Modular self‐assembly of a Y‐shaped multiprotein complex from seven nucleoporins

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its ∼30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p‐C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi‐cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y‐shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y‐shaped assembly to an ∼40 nm long stalk. Taken together, our data document that the Nup84p–Nup133p complex self‐assembles in a modular concept from distinct smaller nucleoporin construction sets.

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