Question

i) Discuss the importin-alpha nuclear import cycle, mechanistically accounting for the key steps of nuclear transport. (Essay style include appropriate diagrams)

ii) Quantitative reasoning, 10 marks: During DNA replication, the genome is duplicated as chromatin. How fast does nuclear transport of histones have to take place in terms of molecules/pore/second in order to support S-phase in human cells? Assume the genome is 6 x 109 bp, a nucleosome spans 200 bp and that 75% of the genome is replicated in the first 3 hr of S-phase. Nuclei contain 2000 nuclear pore complexes. (show work)

Answer 1

Importin α are composed of a flexible N-terminal importin-β-binding (IBB) domain and a highly structured domain comprised of ten tandem armadillo (ARM) repeats . The helical ARM repeats assemble into a twisted slug-like structure, whose belly serves as the cNLS-binding groove. The exportin CAS binds to the tenth ARM repeat. The flexible IBB domain interacts either in trans with importin β or in cis with the cNLS-binding groove. Through these interactions, the IBB domain acts as a competitive inhibitor to regulate binding of cNLS cargo to the binding groove and, when displaced, it enables the binding of the cNLS to the nascent targeting complex. The concentration of basic residues within the IBB domain, which is characteristic of cNLS sequences (see below), and its affinity for the cNLS groove are consistent with the notion that it originally functioned as the NLS for an importin α progenitor, one that began its life as just another cargo for an early importin β. What we do know for certain is that IBB domains have important roles in the control of ternary complex assembly and disassembly.

The formation of the importin-α/β–cNLS cargo ternary complex is the first step in the nuclear transport of hundreds of different nuclear proteins, and, as such, is tightly regulated (Box 1). The N-terminal IBB domain serves a dual role. It binds to importin β to target the complex to the NPC for translocation ,but it also contains an autoinhibitory sequence that mimics a cNLS and regulates binding of cNLS cargo to the ARM domain of importin α When importin α is not bound to importin β, the autoinhibitory sequence within the N-terminal domain apparently interacts with the NLS-binding pocket .This interaction is not exceptionally strong because cNLS cargos can still bind to importin α in the absence of importin β, albeit with significantly lower affinity. This mode of cis-regulation is not unique among ARM domain proteins because the C-terminus of β-catenin regulates its function and interacts by the yeast two-hybrid assay with the ARM repeats of the protein and N-terminal sequences .The order of importin α binding to cNLS cargo and importin β is not known. As shown in Figure, importin α could either interact first with importin β to relieve autoinhibition of the NLS-binding groove and then interact with cNLS cargo, or the cNLS cargo could bind to the NLS-binding groove first, thus liberating the autoinhibitory IBB domain to interact with importin β. The observation that importin β increases the in vitro affinity of importin α for cNLS peptides from the micromolar to the nanomolar range is consistent with either model .It is even possible that the order of binding varies from event to event, or occurs in a concerted fashion, depending on the particular cNLS cargo and the effective concentrations of the relevant factors.

Control of ternary complex assembly and disassembly. (a) Assembly of the ternary complex could occur by two distinct mechanisms, although in vitro affinity measurements support the binding of classical nuclear localization signal cargo to the importin α/β heterodimer. (b) The importin-β-binding (IBB) domain, Nup2p and CAS might promote the release of cargo in the