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.

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