Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

Abstract

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes. INTRODUCTIONIn eukaryotic cells the nucleocytoplasmic transport of most proteins and some RNAs is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (also referred to as importins and exportins; reviewed in Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). After synthesis in the cytoplasm, most nuclear protein cargoes are bound by a member of the karyopherin family, through direct interaction with a nuclear localization sequence contained in the cargo protein. Transport through the nuclear pore complex occurs via transient interactions of the karyopherin with the NPC. Once in the nucleus, the karyopherin encounters a high concentration of RanGTP, which acts as a regulator of transport. Interaction of the karyopherin with RanGTP leads to dissociation of the karyopherin from its nuclear cargo (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In some circumstances, nuclear-binding partners of the cargo appear to also play a role in stimulating the dissociation of Kap and cargo (Senger et al., 1998;Lee and Aitchison, 1999;Pemberton et al., 1999). In yeast, there are 14 members of the karyopherin family, with Ͼ20 members in mammalian cells (Mosammaparast and Pemberton, 2004). Karyopherins appear to function in either nuclear import or nuclear export, with only two examples of a Kap that works in both directions (Weis, 2003;Harel and Forbes, 2004;Mosammaparast and Pemberton, 2004). In yeast, 11 members of the karyopherin family must import at least 1500 nuclear proteins, suggesting that each receptor must have many cargoes. To date specific transport receptor-cargo pairs have only been shown for ϳ30 cargoes; in addition the NLS sequences recognized by most Kaps is n...

Kap104p Imports the PY-NLS-containing Transcription Factor Tfg2p into the Nucleus

“…Kap114p imports TFIIB and the TATA-binding protein (43)(44)(45); Kap119p imports TFIIS and Kap122p imports TFIIA (46,47). These Kap␤s also import proteins other than transcription factors.…”

Nucleocytoplasmic transport in yeast: a few roles for many actors

“…Similarly, Kap121, Kap108 and Kap119 can supplement for the absence of Kap123 [38]; however, complete loss of Kap123 results in growth defect [39], suggesting decreased rates of transport in the absence of Kap123. Kap114 is equipped with multiple cargo-binding domains and is thus able to import several proteins simultaneously [40]. Additionally, nuclear import of histones is linked to various Kaps [41,42].…”

Evolutionary and Transcriptional Analysis of Karyopherin β Superfamily Proteins

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