Screening of amphipathic helices identifies features linked to inner nuclear membrane properties

Inner nuclear membrane (INM) proteins control numerous nuclear functions, yet properties governing peripheral INM-association remain poorly defined. Here, we perform an image-based screen to identify features in candidate and established amphipathic helices (AHs) that mediate INM association. AHs that localize to ER/Golgi membranes become INM-associated when directed to the nucleus, whereas mitochondrial-localized AHs become largely nucleoplasmic. Mutating a mitochondrial-localized AH to increase its preference for membranes with lipid packing defects enables INM-association upon nuclear targeting. Structural studies of an INM-associated AH in TMEM214 show folding upon binding to lipid packing defects, and full-length TMEM214 localizes to nuclear pores. Consistently across multiple AHs, INM binding depends primarily on sensitivity to lipid packing defects, with a minor electrostatic contribution. Highlighting distinct effects of different mechanical inputs, nuclear swelling, but not cell stretching, enhances INM association of select AHs. These findings define AH features that promote INM association, with implications for nucleo-mechanical responses.