An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore

We described previously an assay for authentic nuclear protein import in vitro. In this assay, exogenous nuclei are placed in an extract of Xenopus eggs; a rhodamine-labeled protein possessing a nuclear localization signal is added, and fluorescence microscopy is used to measure nuclear uptake. The requirement in this system for a cytosolic extract suggests that nuclear import is dependent on at least one cytosolic factor. We now confirm this hypothesis. Treatment of the cytosol with N-ethylmaleimide (NEM) abolishes nuclear protein import; readdition of a cytosolic fraction to the NEM-inactivated extract rescues transport. Thus, at least one NEM-sensitive factor required for transport is supplied by the cytosol. This activity, called nuclear import factor-1, or NIF-1, is ammonium-sulfate-precipitable, protease-sensitive, and heat-labile; it is therefore at least partly proteinaceous. NIF-1 stimulates, in a concentration-dependent manner, the rate at which individual nuclei accumulate protein. The effect of NIF-1 is enhanced by a second cytosolic NEM-sensitive factor, NIF-2. Earlier we identified two steps in the nuclear import reaction: (a) ATP-independent binding of a signal-sequence-bearing protein to the nuclear pore; and (b) ATP-dependent translocation of that protein through the pore. We now show that NEM inhibits signal-mediated binding, and that readdition of NIF-1 restores binding. Thus, NIF-1 is required for at least the binding step and does not require ATP for its activity. NIF-1 may act as a cytoplasmic signal receptor that escorts signal-bearing proteins to the pore, or may instead promote signal-mediated binding to the pore in another manner, as discussed.     

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

A nuclear envelope protein linking nuclear pore basket assembly, SUMO protease regulation, and mRNA surveillance

The nuclear pore complex (NPC) is both the major conduit for nucleocytoplasmic trafficking and a platform for organizing macromolecules at the nuclear envelope. We report that yeast Esc1, a non-NPC nuclear envelope protein, is required both for proper assembly of the nuclear basket, a structure extending into the nucleus from the NPC, and for normal NPC localization of the Ulp1 SUMO protease. In esc1Delta cells, Ulp1 and nuclear basket components Nup60 and Mlp1 no longer distribute broadly around the nuclear periphery, but co-localize in a small number of dense-staining perinuclear foci. Loss of Esc1 (or Nup60) alters SUMO conjugate accumulation and enhances ulp1 mutant defects. Similar to previous findings with Mlp1, both Esc1 and Ulp1 help retain unspliced pre-mRNAs in the nucleus. Therefore, these proteins are essential for proper nuclear basket function, which includes mRNA surveillance and regulation of SUMO protein dynamics. The results raise the possibility that NPC-localized protein desumoylation may be a key regulatory event preventing inappropriate pre-mRNA export.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.     

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.     

Interleukin-3 and bryostatin 1 mediate rapid nuclear envelope protein phosphorylation in growth factor-dependent FDC-P1 hematopoietic cells. A possible role for nuclear protein kinase C

Interleukin-3 (IL-3) is a lymphokine which stimulates the proliferation of normal and transformed multilineage hematopoietic cells. Recently we reported that bryostatin 1, a macrocyclic lactone and potent activator of protein kinase C, could stimulate normal multipotential hematopoietic progenitor cells in vitro in the absence of added polypeptide growth factors. We have now used the murine IL-3-dependent cell line FDC-P1, derived from normal murine marrow cells, to examine the early biochemical events associated with stimulation of hematopoietic cells. We find that both IL-3 and bryostatin 1 are mitogenic and stimulate the growth of FDC-P1 cells. Cells grown for extended periods in the presence of bryostatin 1 (1 nM) alone retain IL-3 responsiveness, indicating that bryostatin 1 does not induce an IL-3-independent state. Protein phosphorylation studies in cells treated with either IL-3 or bryostatin 1 indicate that both stimulators can mediate the rapid (within 5 min) serine-specific phosphorylation of several nuclear envelope polypeptides, including lamin B. Both IL-3- and bryostatin 1-mediated nuclear envelope phosphorylation is dose-dependent, occurring at concentrations which are mitogenic to FDC-P1 cells. The extent of nuclear envelope phosphorylation mediated by IL-3 and bryostatin 1 correlates with the mitogenic response. Furthermore, both mitogens mediate the rapid immunologic translocation of protein kinase C to the nuclear envelope where phosphorylation occurs. These data indicate that the early mitogenic signal(s) generated by IL-3 and bryostatin 1 may converge at the level of the nuclear envelope, perhaps through a protein kinase C-like activity which mediates phosphorylation of specific nuclear envelope polypeptides such as lamin B.     

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.     

Phosphatidic acid is the prominent product of endogenous neuronal nuclear lipid phosphorylation, an activity enhanced by sphingosine, linked to phospholipase C and associated with the nuclear envelope.

Using endogenous lipid substrates, assays of lipid phosphorylation indicated that neuronal nuclei had a considerable superiority in phosphatidic acid (PA) formation when compared with homogenates and other subfractions of cerebral cortex. This predominance of neuronal nuclear PA labelling was linked to a sizable pool of nuclear diacylglycerols that expanded significantly with incubation. PA was also the dominant product of neuronal nuclear lipid phosphorylation reactions. Nuclear envelope preparations and the parent neuronal nuclei showed specific rates of PA formation that were comparable, based upon membrane phospholipid contents. As well, using an exogenous diacylglycerol substrate, the distribution of diacylglycerol kinase activities closely followed phospholipid contents of subfractions derived from the neuronal nucleus during envelope preparation. This evidence suggested an association between diacylglycerol kinase and the neuronal nuclear envelope. Nuclear PA formation increased in the presence of sphingosine, while sphingosine decreased PA formation in other subfractions. Likely sphingosine exerted its effect on nuclear diacylglycerol kinase, as sphingosine did not elevate levels of nuclear diacylglycerols. Phosphoinositidase C was present in the nuclei and inhibitors of this enzyme did decrease PA formation, indicating diacylglycerols from inositides as substrates for nuclear diacylglycerol kinase. The nuclear envelope fraction had a considerably lower specific phosphoinositidase C activity than the parent nuclei, and showed an activation of PA formation by sphingosine, but a less efficient handling of the exogenous diacylglycerol substrate. It is possible that phosphoinositidase C and diacylglycerol kinase are closely situated within the neuronal nuclei, and a loss of the former activity may compromise the latter.     

Nuclear envelope fraction proteins: Isolation and comparison with the nuclear protein of the avian erythrocyte

A nuclear envelope fraction is obtained from circulating erythrocytes of mature chickens. This fraction was characterized by chemical analysis, ultramicroscopic examination and electrophoretic analysis of the polypeptides. It is free of plasma membrane polypeptides, of hemoglobin and of deoxyribonucleohistone. Comparisons of the sodium dodecyl sulfate-polyacrylamide-gel electropherograms of the nuclear envelope fraction polypeptides with either chromosomal fraction or the total nonhistone fraction polypeptides from Triton X-100-washed nuclei indicate that certain major polypeptides are common to all fractions. This indicates that many chicken erythrocyte nonhistone “chromosomal” polypeptides may be primarily associated with nuclear structural elements which are not, in fact, deoxyribonucleohistone. Although this finding cannot be extended per se to other cells, it does suggest that caution should be exercised in describing nuclear components as “chromosomal” when this designation is intended to imply primary association with the deoxyribonucleohistone. This analysis of the polypeptides in the nuclear envelope fraction of the erythrocyte indicates that further study of the envelope fraction will provide new insights into nuclear structure and function.     

Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope.

The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.     

Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.     

Sequence and characterization of cytoplasmic nuclear protein import factor p97

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.     

Dynamic associations of heterochromatin protein 1 with the nuclear envelope

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.     

Potentiation of a sodium-calcium exchanger in the nuclear envelope by nuclear GM1 ganglioside

Calcium is recognized as an important intracellular messenger with a pivotal role in the regulation of many cytosolic and nuclear processes. Gangliosides of various types, especially GM1, are known to have a role in some aspects of Ca2+ regulation, operating through a variety of mechanisms that are gradually coming to light. The present study provides evidence for a sodium-calcium exchanger in the nuclear envelope of NG108-15 neuroblastoma cells that is potently and specifically activated by GM1. Immunoblot analysis revealed an unusually tight association of GM1 with the exchanger in the nuclear envelope but not with that in the plasma membrane. Exchanger and associated GM1 were located in the inner membrane of the nuclear envelope, suggesting this system could function to transfer Ca2+ between nucleoplasm and the envelope lumen. The GM1-enhanced exchange was blocked by cholera toxin B subunit while C2-ceramide, a recently discovered inhibitor of the exchanger, blocked all transfer. Exchanger activity was significantly elevated in nuclei isolated from cells that were induced to differentiate by KCl + dibutyryl-cAMP, a treatment previously shown to promote up-regulation of nuclear GM1 in conjunction with axonogenesis. Similar enhancement was achieved by addition of exogenous GM1 to nuclei from undifferentiated cells. These results suggest a prominent role for nuclear GM1 in regulation of nuclear Ca2+ homeostasis.     

A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport.

Hsp70 has been implicated in nuclear localization signal (NLS)-directed nuclear transport. Saccharomyces cerevisiae contains distinct SSA and SSB gene families of cytosolic Hsp70s. The nucleocytoplasmic localization of Ssa1p and Ssb1p was investigated using green fluorescent protein (GFP) fusions. Whereas GFP-Ssa1p localized both to the nucleus and cytoplasm, GFP-Ssb1p appeared only in the cytosol. The C-terminal domain of Ssb1p contains a leucine-rich nuclear export signal (NES) that is necessary and sufficient to direct nuclear export. The accumulation of GFP-Ssb1p in the nuclei of xpo1-1 cells suggests that Ssb1p shuttles across the nuclear envelope. Elevated levels of SSA1 but not SSB1 suppressed the NLS-GFP nuclear localization defects of nup188-Delta cells. Studies with Ssa1p/Ssb1p chimeras revealed that the Ssb1p NES is sufficient and necessary to inhibit the function of Ssa- or Ssb-type Hsp70s in nuclear transport. Thus, NES-less Ssb1p stimulates nuclear transport in nup188-Delta cells and NES-containing Ssa1p does not. We conclude that the differential function of Ssa1p and Ssb1p in nuclear transport is due to the NES-directed export of the Ssb1p and not to functional differences in their ATPase or peptide binding domains.     

Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells

Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed.     

Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures

Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.     

Electrostatic association of glutathione transferase to the nuclear membrane. Evidence of an enzyme defense barrier at the nuclear envelope

The possible nuclear compartmentalization of glutathione S-transferase (GST) isoenzymes has been the subject of contradictory reports. The discovery that the dinitrosyl-diglutathionyl-iron complex binds tightly to Alpha class GSTs in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M., and Ricci, G. (2007) J. Biol. Chem. 282, 6364-6371) prompted us to reconsider the nuclear localization of GSTs in these cells. Surprisingly, we found that a considerable amount of GSTs corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2, and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy, immunofluorescence experiments, and molecular modeling have been used to detail the electrostatic association in hepatocytes and liposomes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent an amazing novelty in cell physiology. The interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intranuclear localization of Alpha GSTs.     

Comparison of cytosolic and nuclear poly(A) polymerases from rat liver and a hepatoma: structural and immunological properties and response to NI-type protein kinases

Poly(A) polymerases were purified from the cytosol fraction of rat liver and Morris hepatoma 3924A and compared to previously purified nuclear poly(A) polymerases. Chromatographic fractionation of the hepatoma cytosol on a DEAE-Sephadex column yielded approximately 5 times as much poly(A) polymerase as was obtained from fractionation of the liver cytosol. Hepatoma cytosol contained a single poly(A) polymerase species [48 kilodaltons (kDa)] which was indistinguishable from the hepatoma nuclear enzyme (48 kDa) on the basis of CNBr cleavage maps. Liver cytosol contained two poly(A) polymerase species (40 and 48 kDa). The CNBr cleavage patterns of these two enzymes were distinct from each other. However, the cleavage pattern of the 40-kDa enzyme was similar to that of the major liver nuclear poly(A) polymerase (36 kDa), and approximately three-fourths of the peptide fragments derived from the 48-kDa species were identical with those from the hepatoma enzymes (48 kDa). NI-type protein kinases from liver or hepatoma stimulated hepatoma nuclear and cytosolic poly(A) polymerases 4-6-fold. In contrast, the liver cytosolic 40- and 48-kDa poly(A) polymerases were stimulated only slightly or inhibited by similar units of the protein kinases. Antibodies produced in rabbits against purified hepatoma nuclear poly(A) polymerase reacted equally well with hepatoma nuclear and cytosolic enzyme but only 80% as well with the liver cytosolic 48-kDa poly(A) polymerase and not at all with liver cytosolic 40-kDa or nuclear 36-kDa enzymes. Anti-poly(A) polymerase antibodies present in the serum of a hepatoma-bearing rat reacted with hepatoma nuclear and cytosolic poly(A) polymerases to the same extent but only 40% as well with the liver cytosolic 48-kDa enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)