The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors

Abstract

Transport receptors of the importin β family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin β involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin β also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin β. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin β. These effects of hsc70 were observed in the nuclear export of importin β, but also for other import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

Nucleocytoplasmic Distribution Is Required for Activation of Resistance by the Potato NB-LRR Receptor Rx1 and Is Balanced by Its Functional Domains

“…SGT1 has been found in the cytoplasm and also in the nucleus (Noë l et al, 2007) and interacts with chaperones that have been implicated in assisting nuclear shuttling, like HSP90 or HSC70 (Tago et al, 2004;Kose et al, 2005;Brkljacic et al, 2009;Cazalé et al, 2009). We tested whether the absence of SGT1 would affect the localization of Rx1.…”

“…For the nuclear localization, it requires a complete SGS domain, which functions as interaction domain for HSC70. The chaperone HSC70 can actively be transported to the nucleus via an NLS and has been shown to play a role in nucleocytoplasmic shuttling in several eukaryotic systems (Kose et al, 2005;Cazalé et al, 2009). Similarly, HSP90 plays a role in nucleocytoplasmic shuttling of, among others, metazoan nuclear receptors like the glucocorticoid receptor (Kang et al, 1994;Tago et al, 2004;Echeverría et al, 2009).…”

70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY

“…The heat shock cognate protein hsc70 has been previously reported to play a role in modulating the nuclear import of proteins such as SV40-T-ag and nucleoplasmin (9, 10), as well as being a CaM-binding protein (21,22) able to associate with nucleoporins (7). As a first step in establishing the potential role of hsc70 in CaM-dependent SRY nuclear import, we employed an established hsc70 in SRY Nuclear Import…”

“…HeLa cells were transfected using siPORT TM NeoFX TM (Ambion) or Lipofectamine TM 2000 (Invitrogen) for siRNA (100 nM hsc70 Stealth siRNA duplexes (Invitrogen) with the sense sequence 5Ј-UAAUUCUAAGUACAUUGAGACCAGC-3Ј) (7) or plasmid DNA expression constructs respectively, according to the manufacturer's specifications. Cells were imaged live by confocal laser scanning microscopy (CLSM, Nikon C1) 20h posttransfection and quantitative analysis of digitised images performed using Image J (National Institutes of Health, Bethesda, MD) for the nuclear (Fn) and cytoplasmic (Fc) fluorescence subsequent to subtraction of autofluorescence to derive the nuclear to cytoplasmic ratio (Fn/c) as described previously (3,15).…”

“…The 70-kDa heat shock cognate protein hsc70 has been previously shown to be involved in modulating nuclear transport, with a role in facilitating the subcellular localization of members of the Imp family (7), as well as in regulating the nuclear * This work was supported by Australian Research Council Grant COE348239…”