Abstract
Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta. The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha 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 alpha remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm.
Highlights
In eukaryotes, the nuclear envelope provides an essential barrier that separates the nuclear genome from the intermediary metabolism, signaling systems, and translation machinery of the cytoplasm
Over the last several years many studies have led to a detailed model for the individual steps in the classic nuclear transport cycle [5, 16]: 1) importin ␣ binds to the nuclear localization signal (NLS)-cargo to form a trimeric import complex with importin ; 2) this NLS-cargo/importin ␣/importin  complex is targeted to the nuclear pore complexes (NPC) by importin ; 3) the complex translocates into the nucleus where it encounters RanGTP; 4) upon binding RanGTP, importin  dissociates from NLS-cargo/importin ␣; 5) NLS-cargo is released from importin ␣; and 6) once cargo is released, importin ␣ is recycled to the cytoplasm by its export receptor, Cse1p/CAS, in a trimeric complex with RanGTP
This study demonstrates that the N-terminal IBB domain of importin ␣ has two essential functions in vivo
Summary
Plasmids, and Chemicals—All chemicals were obtained from Sigma or USBiological unless otherwise noted. The dissociation constants for the binding of SV40 NLS-GFP and IBB-GFP to importin ␣ were measured essentially as described previously [26, 38]. The assay was carried out as described above except changes in the anisotropy of IBB-GFP were monitored in the presence of increasing amounts of importin . This yielded a dissociation constant for IBB-GFP binding to importin . The Kd values for wild-type and mutant importin ␣ proteins binding to importin  were determined by fitting the resulting binding curves to an equation for the fraction of IBB-GFP
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