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 Targeting of Endoplasmic Reticulum (ER)-synthesized membrane proteins to the Inner Nuclear Membrane (INM) has long been explained by the ‘diffusion-retention model’. However, several INM proteins contain non-classical Nuclear Localization Signal (NLS) sequences that, in a few instances, have been shown to promote importin α/β– and Ran–dependent translocation to the INM. Here, using structural and biochemical methods, we show that yeast INM proteins Heh2 and Src1/Heh1 contain bipartite import sequences that associate intimately with the minor NLS-binding pocket of yeast importin α and unlike classical NLSs efficiently displace the IBB-domain in the absence of importin β. In vivo, the intimate interactions at the minor NLS-binding pocket make the h2NLS highly efficient at recruiting importin α at the ER and drive INM localization of endogenous Heh2. Thus, h1/h2NLSs delineate a novel class of super-potent, IBB-like membrane protein NLSs, distinct from classical NLSs found in soluble cargos, and of general interest in biology.

Highlights d Inner nuclear membrane proteins Heh1 and Heh2 have unique sorting signals d Their sorting signals are composed of a long intrinsically disordered linker and NLS d Linkers that best support import are flexible and match dimensions of NPC scaffold