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Conclusion: hsc70 is a key mediator of the Ca M-dependent nuclear transport mechanism of SRY.Significance: hsc70 may mediate nuclear transport of other developmentally important transcription factors.
Here, we show, for the first time, that the 70-k Da heat shock cognate protein hsc70 plays a key role in Ca M-dependent nuclear import of SRY.
Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain Ca M-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both Ca M and hsc70, in contrast to an SRY mutant derivative with impaired Ca M binding.
However, little is known of the mechanism by which Ca M facilitates nuclear translocation.
The 70-k Da 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 transport of proteins such as the temperature sensitive p53 mutant p53 (8), Simian virus 40 large tumor antigen (T-ag) (9) and nucleoplasmin (10).
Experiments were carried out in 5 μl containing 10 μ GFP-fusion protein, a 70-k Da Texas red dextran to assess nuclear integrity, untreated rabbit reticulocyte lysate (45 μg/μl; Promega, Madison, WI), and an ATP regenerating system (0.125 g/ml creatine phosphokinase; 30 m hsc70 (Stressgen Biotechnologies) and/or Ca M (Calbiochem, La Jolla, CA) purified protein.