Evidence for Distinct Substrate Specificities of Importin α Family Members in Nuclear Protein Import

Abstract

Importin ␣ plays a pivotal role in the classical nuclear protein import pathway. Importin ␣ shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin ␤-dependent import pathway. In contrast to what is found for importin ␤, several isoforms of importin ␣, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin ␣7. To analyze specific functions of the distinct importin ␣ proteins, we recombinantly expressed and purified five human importin ␣'s along with importin ␣ from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin ␣ proteins from humans or Xenopus bind their import receptor (importin ␤) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin ␣ proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin ␣3, whereas most of the other substrates were imported by all importin ␣ proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin ␣ proteins were revealed when two substrates were offered simultaneously.

AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1

“…Second, as in the study by Gü ttinger et al, importin ␣5 did not appear to bind to HuR, and according to our data, neither did other ␣ importins except importin ␣1. In this regard, other proteins like RCC1, STAT-1, and STAT-2 are known to be imported via single ␣ importin isoforms (44,(62)(63)(64). Finally, it remains to be assessed whether a lack of Kap␤2 in living cells results in a transport defect for HuR in vivo (36).…”

“…Anti-HA antibody was from Qiagen. Polyclonal antibodies recognizing importins ␣1, ␣3, ␣4, ␣5, and ␣7 were previously described (44). Following secondary antibody incubations, signals were detected by enhanced chemiluminescence.…”

Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

“…Importin ␣ molecules serve as adapters that bind directly to the NLS and link NLS-containing proteins to importin ␤, which subsequently docks the ternary complex at the cytoplasmic side of the NPC and mediates its translocation into the nucleus (Goldfarb et al, 2004). Six importin ␣ paralogs that show different affinity to particular substrates exist in humans (Kohler et al, 1999). The paralogs are classified into three subfamilies based on sequence similarity: P, importin ␣1; Q, importin ␣3 and 4; and S, importin ␣5, 6, and 7.…”

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

“…Expression plasmids for GFP fusions of human histones H3 and H2b, and thyroid hormone receptor b (TR), were provided by Dr. Hiroshi Kimura, Tokyo Medical and Dental University (18), and Dr. Padma Maruvada, National Institutes of Health, Bethesda, respectively (19). The cDNA of human importin-a5 (provided by Matthias Köhler; Reha-Klinik Damp, Damp) (20) was amplified by PCR and cloned into the EcoRI and XhoI sites of pASK-IBA3plus (IBA, Göttingen). A plasmid encoding Histagged human importin-b was provided by Dr. Dirk Görlich, MPI für Biophysikalische Chemie, Göttingen (21).…”