Biomedical subjects
Publications and source records attributed to W Deppert.
We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.
Infection of cells with adenovirus lead to a characteristic reorganization of all cytoskeleton systems, starting with alterations at the microtubuli of the cells. During this progress, the flat, extended, and polar morphology of the cytoskeleton became nonpolar and rounder. These rearrangements were initiated before the appearance of adenovirus structural proteins hexon and fiber, as well as before the shutoff of host protein synthesis. We conclude that these alterations reflect a specific reorganization rather than an unorganized breakdown of the cell during adenovirus infection.
To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T.
Cells of Dictyostelium discoideum were incubated with [H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane-associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti-actin antibody, its coincidence with the major [S]methionine-labeled protein after two-dimensional electrophoresis and its binding to a DNase I affinity column. The H-label was resistant to chloroform-methanol extraction and boiling in SDS-containing buffer. After partial purification by preparative SDS-polyacrylamide gel electrophoresis, the 44-kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas-liquid chromatography.
In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.
Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40-specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses.
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Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies.
To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.
HeLa cells infected with the adenovirus 2-simian virus 40 (Ad2+SV40) hybrid virus Ad2+ND2 were labeled with either [35S]methionine or [3H]palmitate and fractionated into cytoplasmic, nuclear, and plasma membrane fractions. Analysis of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SV40-specific proteins in the plasma membrane fraction were specificially acylated.
SV40 transformed mouse cells (mKSA) were labeled in parallel with either [35S]methionine or [3H]palmitate and subfractionated. Nuclear extracts and solubilized plasma membranes were analyzed for the presence of either 35S- or 3H-labeled SV40 large tumor antigen by immunoprecipitation and SDS polyacrylamide gel electrophoresis. The majority of the [35S]methionine labeled large T was recovered from the nuclear fraction, only minor amounts were detected in plasma membranes. In contrast, large T labeled specifically with [3H]palmitate was found only in the plasma membrane fraction. Our results demonstrate a specific acylation of large T associated with plasma membranes, suggesting that the membrane location of this predominantly nuclear protein is specific.
Analysis of cellular extracts of HeLa cells infected with adenovirus type 2 (Ad2) by immunoprecipitation with antiserum against the late nonstructural 100,000-dalton (100K) protein revealed the presence of a specific complex between the 100K protein and newly synthesized hexon molecules. Serological analysis of the hexon molecule in the 100K/hexon complex with antibodies specific for hexon monomers or trimers showed that only monomeric hexon molecules were associated with the 100K protein. By immunofluorescence microscopy this monomeric hexon was primarily found in the cytoplasm, whereas the trimeric form was mainly confined to the nucleus of infected cells. We conclude that in the cytoplasm of Ad2-infected cells newly synthesized, monomeric hexon molecules can interact with the 100K protein. This suggests that the 100K protein may play some role either in trimerization of newly synthesized, monomeric hexon molecules and/or in its transport from the cytoplasm into the nucleus.
We have analyzed the plasma membrane association of the SV40 large tumor antigen (large T) in SV40-transformed BALB/c mouse tumor cells (mKSA). Isolated plasma membranes were subfractionated: treatment with the non-ionic detergent Nonidet P40 (NP40) resulted in a NP40-resistant plasma membrane lamina, which could be further extracted with the zwitterionic detergent Empigen BB. Analysis of the different plasma membrane fractions revealed that only about one third of large T associated with isolated plasma membranes could be solubilized with NP40. The residual plasma membrane-associated large T was tightly bound to the NP40-resistant lamina of the plasma membrane from which it was released by treatment with the zwitterionic detergent Empigen BB. Further evidence for a specific interaction of a distinct subclass of large T with the plasma membrane was provided by showing that only T associated with the NP40-resistant lamina of the plasma membrane contained covalently bound fatty acid. Neither nuclear large T nor large T in the NP40-soluble plasma membrane fraction could be labeled with [3H]palmitic acid. Our results indicate that an acylated subclass of large T interacts specifically with a structure of the plasma membrane, suggesting that it might be involved in a membrane-dependent biological function.
To define the interaction of SV40 large T with different structural systems in the nuclei of SV40-transformed cells (BALB/c mKSA), we have employed an in situ cell fractionation procedure leading to the preparation of the nuclear matrix, and giving rise to defined nuclear extracts comprising soluble nuclear proteins (nucleoplasm) and the solubilized chromatin. Large T could be detected in the nucleoplasmic fraction and in the chromatin fraction, as well as in tight association with the nuclear matrix. From the nuclear matrix, large T could be solubilized by treatment with a zwitterionic detergent. Different solubility properties, differences in the amount of the cellular phosphoprotein p53 coprecipitating with large T, and different stabilities in its association with the nuclear structural systems indicate that distinct subclasses of large T were isolated from their in vivo location in SV40-transformed cells.
The major polypeptides of nuclear matrix preparations from vertebrate tissues are rather similar. However, nuclear matrix fractions isolated from cultured vertebrate cells of different origin show variations in their major polypeptides. We demonstrate that cytoplasmic intermediate filaments copurify with nuclear matrices from these cells. Because of their abundance, their subunit proteins form major bands on SDS-polyacrylamide gels. The tissue specificity of the intermediate filament proteins then gives rise to the variations observed. To explain the differences in major proteins of nuclear matrices isolated from vertebrate cells grown in tissue and in culture we have analyzed the distribution of intermediate filaments during isolation of nuclei from liver tissue. We show that during homogenization of liver intermediate filaments are torn off and can be separated from the nuclei. Nuclear matrix preparations from these nuclei, therefore, do not contain intermediate filaments and true nuclear matrix proteins (e.g. lamins) are the major protein species. Our results suggest that the major nuclear matrix polypeptides (lamins) are similar in all vertebrate cells, since lamin like proteins were identified in cultured cells, too. Using antisera we demonstrate an immunological difference between lamins A/C and lamin B.
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