The auto-inhibitory function of importin α is essential in vivo

Abstract

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin α and β. The importin α subunit recognizes classical NLS sequences, and the importin β subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin β binding (IBB) domain of importin α regulates NLS-cargo binding in the absence of importin β in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin α protein. These mutants dissect the two functions of the N-terminal IBB domain, importin β binding and auto-inhibition. One of these importin α mutations, A3, decreases auto-inhibitory function without impacting binding to importin β or the importin α export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin α remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin α is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin α to the cytoplasm.