Studies on the Molecular Mechanism of the Translocation of Estrogen Receptor from the Cytoplasm into the Nucleus12

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Abstract
A detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER.E (basic ER molecule bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes [“5S” ER.E, (vero-ER.E).(component A); “6S” ER.E, (vero-ER.E).(component B)6; “8S” ER.E, (vero-ER.E).(component B)6.(component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER.E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with “5S” ER.E under low salt conditions was indicated. The complex, (“5S” ER.E).(component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei. It was assumed that this adsorption of (“5S” ER.E).(component Q to the nuclear envelopes was taken as translocation into the nucleus in the previous studies. The ER-fragments [secto-ER.E (sedimentation coefficient, 4.5S; Stokes radius, 35 A) and “3.8S” ER.E (sedimentation coefficient, 3.8S; Stokes radius, 32 A)] obtained by the proteolysis of vero-ER.E by the endogenous proteases, in which the binding sites to ERBFs are destroyed, did not translocate into the nuclei. These results indicated that the binding site (or the adjacent region) of vero-ER to ERBFs plays an indispensable role in the translocation of vero-ER.E from the cytoplasm into the nucleus.