The roles of multiple importins for nuclear import of murine aristaless-related homeobox protein.

Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.

Subunit-subunit interactions in the human 26S proteasome.

Ubiquitin-dependent proteolysis is mediated by the proteasome. To understand the structure and function of the human 26S proteasome, we cloned complete ORFs of 32 human proteasome subunits and conducted a yeast two-hybrid analysis of their interactions with each other. We observed that there are 114 interacting-pairs in the human 26S proteasome. About 10% (11/114) of these interacting-pairs was confirmed by the GST-pull down analysis. Among these observed interacting subunits, 58% (66/114) are novel and the rest 42% (48/114) has been reported previously in human or in other species. We observed new interactions between the 19S regulatory particle and the beta-rings of the 20S catalytic particle and therefore proposed a modified model of the 26S proteasome.

Interaction between importin 13 and myopodin suggests a nuclear import pathway for myopodin.

Importin 13 is a member of the importin beta superfamily of nuclear transport proteins and is expressed in multiple tissues at high levels both in humans and rodents, including fetal lung, brain, and heart. In order to elucidate potential functions of imp13 in the heart, we have used rat imp13 as bait to screen a human heart cDNA library and identified an interaction with the C-terminal peptide of myopodin (a.a. 360-698), an actin-bundling protein, associated with tumor-suppressor activity that localizes to both the cytoplasm and the nucleus. We have used GST-pull down assays and co-immunoprecipitation experiments to demonstrate an interaction between imp13 and full-length myopodin and observed that RanGTP dissociates the myopodin-imp13 complex. In studies of cultured cells, we show that both imp13 siRNA and a C-terminal fragment of imp13 protein prevent nuclear localization of myopodin. We, therefore, conclude that imp13 functions in myopodin import and we suggest that the regulation of these events is critical for normal and abnormal cellular differentiation.