Discrimination in the uptake of soluble proteins by isolated nuclei

Abstract

ABSTRACT The uptake of radioactive cytosol proteins has been studied in isolated HeLa cell nuclei. The process is temperature- and pH-dependent, is enhanced by divalent metal ions, and is saturable with respect to the protein concentration of the cytosol. Nuclei prepared by washing in either detergent or sucrose buffers are similar in their abilities to accumulate protein, indicating that the outer nuclear membrane does not affect the process. The uptake is neither inhibited to any great extent by cyanide or 2,4-dinitrophenol nor significantly stimulated by glucose, glucose 6-phosphate, D-lactate, succinate, L-glutamate, NADH, phosphoenolpyruvate, cyclic AMP, or ATP. In fact, ATP is inhibitory, apparently due to its ability to chelate magnesium ions. Fractionation of the cytosol preparations on DEAE-cellulose shows that, as a class, those proteins eluting at higher salt concentrations are taken up better than those eluting at lower salt concentrations. Incubation of nuclei with protein fractions obtained from the chromatography of cytosol extracts on Sephadex G-150 indicates that the uptake of proteins in the molecular weight range from 35000 to 145000 is inversely proportional to molecular weight, but the nuclear accumulation of very small proteins (Mr 15000) was less than would be expected if molecular weight were the only factor determining nuclear uptake. Polyacrylamide gel electrophoresis shows that several protein classes in the cytosol are taken up by the nucleus and that in some cases the amount of protein in the nucleus reflects its relative concentration in the cytosol. Exceptions, however, are the drastic reduction in the nuclei of a class of proteins with approximate molecular weights of 55000 to 70000, and the increase in the nuclei of a protein class in the molecular weight range from 30000 to 40000. Proteins released from the nucleus are taken up by nuclei in vitro more efficiently than are cytosol proteins. In the presence of excess nuclei only 5-10 % of the available protein is taken up. Purified histones are rapidly taken up by nuclei, bovine serum albumin is not accumulated at all, and actin is taken up at an intermediate rate. When nuclei are fractionated following incubation, ribosomal proteins are found associated with the nucleolus to a greater extent than are cytosolic proteins. Thus, the nuclear uptake of soluble proteins in this cell-free system exhibits characteristics and specificity similar to that previously reported for in vivo systems and suggests that isolated nuclei may be useful for studying various aspects of nucleus-cytoplasm interaction.