Mechanism for the selection of nuclear polypeptides in Xenopus oocytes. II. Two-dimensional gel analysis.

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Abstract
The role of the nuclear envelope in controlling intracellular protein exchanges was investigated in vivo, by determining the effect of altering nuclear permeability on the protein composition of the nucleoplasm and the nuclear uptake rates of specific endogenous proteins. The nuclear envelopes were disrupted by puncturing oocytes in the region of the germinal vesicle by use of glass needles. Nuclear proteins were analyzed in punctured and control cells by 2-dimensional gel electrophoresis, fluorography and double-labeling techniques. Over 300 nuclear polypeptides were identified in the fluorographs; of these only .apprx. 10-15 varied between punctured and control nuclei; different polypeptides varied in each experiment. These qualitative studies indicate that specific binding within the nucleoplasm, and not selection by the envelope, is the main factor in maintaining the protein composition of the nucleus. The nuclear uptake rates of 5 individual polypeptides, ranging in MW from 43,000 to 100,000, were analyzed by use of double-labeling procedures. Only 1 of the polypeptides (actin) entered the nuclei more rapidly after disruption of the envelope. That the nuclear uptake of certain endogenous proteins is unaffected by puncturing demonstrates that passage across the envelope is not a rate-limiting step in the nucleocytoplasmic exchange of these molecules.