Active nuclear import of soluble cargo involves transport factors that shuttle cargo through the nuclear pore complex (NPC) by binding to phenylalanine-glycine (FG) domains. How nuclear membrane proteins cross through the NPC to reach the inner membrane is presently unclear. We found that at least a 120-residue-long intrinsically disordered linker was required for the import of membrane proteins carrying a nuclear localization signal for the transport factor karyopherin-α. We propose an import mechanism for membrane proteins in which an unfolded linker slices through the NPC scaffold to enable binding between the transport factor and the FG domains in the center of the NPC.

Summary Nuclear pore complexes (NPCs) are ~100 MDa transport channels assembled from multiple copies of ~30 nucleoporins (Nups). One third of these Nups contain phenylalanine-glycine (FG)-rich repeats forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG-repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG-repeats directly bind to multiple scaffold Nups in vitro and act as NPC targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a non-essential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG-repeats as velcro to link NPC subcomplexes, and thus add a new layer of connections to current models of the NPC architecture.

Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER)-resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker's yeast. We asked the question what structure in Ist2 bridges the up to 30 nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM is predicted to be intrinsically disordered (ID). In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to that in Ist2. We found that the localization of both Ist2 and Ssy1 at the cell periphery depends on the presence of a PM-binding domain, an ID linker region of sufficient length and a transmembrane domain that most probably resides in the ER. We show for the first time that an ID amino acid domain bridges adjacent heterologous membranes. The length and flexibility of ID domains make them uniquely eligible for spanning large distances, and we suggest that this domain structure occurs more frequently in proteins that mediate the formation of membrane contact sites.

This study examines whether active transport to the inner nuclear membrane, as shown for yeast membrane proteins Heh1 and Heh2, is conserved in metazoans. In support of this, the nuclear localization signal of metazoan Pom121 shares biochemical, structural, and functional properties with those of Heh1 and Heh2, and a Heh2-derived reporter protein targets to the inner membrane in Hek293T cells.