Distribution of Proteins between Nucleus and Cytoplasm of Amoeba proteus

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distribution . We identified -130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear . About 95% of the other proteins (designated CP, for cytoplasmic proteins) are roughly equally concentrated in nucleus and cytoplasm, but-because the cytoplasm is 50 times larger than the nucleus-about 98% of each of the latter is in the cytoplasm . Of the CP, roughly 5% are not detectable in the nucleus . Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments . Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus . Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed . Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained . Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low . These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm . New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect . Moreover, we show that cytoplasmic "contamination" of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments . We speculate that no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm's large volume ensures that CP will be abundant there . Extending Bonner's idea of "quasi-functional nuclear binding sites" for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand . The other NP (those completely absent from cytoplasm) presumably always are bound with high affinity at their primary functional sites . Fundamental to a full appreciation of interactions between nucleus and cytoplasm in the control of gene expression and other activities of the nucleus is identification of those protein 516 molecules that may be uniquely present in each cell compartment and hence putative candidates for agents responsible for functions restricted to nucleus or cytoplasm. The movement, THE JOURNAL OF CELL BIOLOGY " VOLUME 88 MARCH 1981 516-525 ©The Rockefeller University Press " 0021-9525/81/03/0516/10 $1 .00 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom or lack thereof, of these molecules between the two compartments may also provide clues to possible roles in gene expression or replication. Major, but not exclusive, attention in these matters has focused on nonhistone proteins of the nucleus because of the current consensus that these proteins are the molecules primarily responsible for the specificity of differential expression of genes in different cells or under varying conditions (see, for example, reference 1) . An interesting assortment of methods have been employed to study the distribution of proteins between nucleus and cytoplasm (see Discussion), but until recently only in a few studies have specific intracellular proteins been identified and their distribution characterized. In previous studies, molecules not normally present in cells have been examined ; proteins have been characterized by homogenizing and fractionation of cells ; molecules have been extracted, purified, and reinjected into cells; only one or a few kinds of proteins could be studied; etc. (see reference 2 for review). Most studies centered on the movement (or nonmovement) of molecules from cytoplasm to nucleus; rarely was the movement of molecules from nucleus to cytoplasm or their true intracellular distribution determined with confidence. As a result, these studies provided little advance in understanding of how these molecules function. The particular advantage of our method of study is that we are, in a sense, looking at the full spectrum ofproteins normally present in the nucleus and cytoplasm and examining the behavior and localization of these proteins without ever isolating any cell part or molecule before the final assay. Moreover, in all crucial experiments, that final assay does not require cell lysis; the proteins to be characterized are extracted directly from lyophilized cells . Our fundamental approach is to exchange, by micrománipulation, nuclei between [ 35S]methionine-labeled and unlabeled cells, so that one cell has a labeled protein nucleus in unlabeled cytoplasm and another has the opposite arrangement . We can determine (by further nuclear transplantation if necessary) which proteins are retained by each compartment and which move from one compartment to the other. We have shown, without necessarily identifying particular proteins, that the pattern of protein distribution may reveal how some proteins maycome to serve regulatory roles . Amajor conclusion of this work is that almost all cellular polypeptides can enter the nucleus, but only a fraction of them is retained within the nucleus at a higher concentration than in the cytoplasm. Whereas many ofthe latter polypeptides are present in the cytoplasm at very low concentration, some appear to be entirely absent from the cytoplasm-and only a relatively small proportion of all the protein species in the cytoplasm are undetectable in the nucleus. In the Discussion we consider how the data from several other laboratories on different experimental systems are consistent with these conclusions. The mechanisms by which nuclear, but not cytoplasmic, proteins are retained within the nucleus is not known, although it may be significant that even isolated nuclei show the same kind of discriminations . We propose that whatever is responsible for that selectivity is related to mechanisms responsible for differential gene expression and general changes in nuclear activity . MATERIALS AND METHODS Cells and Cell Culturing Used throughout this work was the free-living ameba, Amoeba proteus, cultured as described earlier (3) . Radioactive Labeling of Cells Amebas were labeled by feeding them ['Slmethionine-labeled tetrahymenas (4). The cells were labeled with 3H-amino acids as follows. Tetrahymenas from a log-phase culturegrowing on 296 proteose peptonewere inoculated into a defined medium (5) in which 150WCi/ml of [3Ii)leucine, 50WCi/ml of [3Nlysine, and 150 pCi/ml of [ 3H)alanine were substituted for their unlabeled analogues and incubated for about 24 h at 29°C . Amebas were fed for 2 d with the labeled tetrahymenas washed free of the defined medium, and then fed unlabeled tetrahymenas for another 2 d. At the end of that period, the amebas averaged 7.5-10 x 10' dpm/cell . Assay of Radioactivity Radioactivity was assayed as described previously (6) . Nuclear Transplantation Nuclei were transplanted according to the method ofJeon and Lorch(7), with the following modifications. To mini,ni~r the adsorption by one cell of any labeled material that might have been released from another cell during nuclear transplantation, the operations were performed in a cell extract made by homogenizing 1 part by volume ofamebas in 3.3 parts by volume ameba medium and removing allparticulate matter by centrifugation. This "operation medium" was roughly equivalent to a 1% protein solution . In some instances, to facilitate a closer examination of the nucleus being transplanted and to exclude any nuclei having any adhering cytoplasm, the nuclei being transplanted were allowed to remain in the external medium for 10 s rather than being pushed directly from one cell into the other. We determined that for --80% of the nuclei this extracellular exposure had no effect on the subsequent survival and reproduction of the recipient cell. For some experiments it was necessary during the transplantation operations to distinguish between two different nuclei in one cell. This was accomplished by arranging for the two nuclei to differ in size (6).