Studies of asymmetric membrane assembly.

The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large (greater than 6000 A diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed.

Cytotoxicity of myeloma light chains in cultured human kidney proximal tubule cells.

We evaluated the effect of eight species of light chains on cultured human kidney proximal tubule cell proliferation. Exposure to light chains for 48 hours caused dose-dependent inhibition in tritium ((3)H)-thymidine incorporation by simian virus 40 immortalized human proximal tubule cells, although the effect was variable among different species of light chains. We studied cytotoxic effects of selected toxic light chains in further detail. Two of these light chains caused significant DNA degradation. A lambda-light chain caused lactate dehydrogenase release from exposed cells at 48 hours, but not at 24 hours. Cytomorphological and electron microscopic examination of cells exposed to light chains for 24 hours showed condensed nuclei, cell detachment, paucity of mitotic activity, and apoptosis, and at 48 hours of exposure, changes consistent with necrosis. Apoptosis assay by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method showed a sixfold increase in the number of apoptotic cells exposed to the same lambda-light chain for 24 hours. Rhodamine-phalloidin staining showed variable but significant disruptions in the actin cytoskeleton. These studies show that some myeloma light chains are toxic to cultured human proximal tubule cells and induce cytoskeletal injury and DNA damage consistent with apoptosis followed by secondary necrosis. Direct proximal tubule cell toxicity may be an important mechanism of renal involvement in multiple myeloma.

The purification of M13 procoat, a membrane protein precursor.

Many membrane proteins and most secreted proteins are initially made as precursors with an N-terminal leader sequence. We now report the isolation of M13 procoat, the precursor of the membrane-bound form of M13 coat protein. There are 40 000 copies of M13 procoat protein/cell during M13 amber 7 virus infection. Purified procoat is quantitatively cleaved by isolated leader peptidase to yield mature-length coat protein. Rabbit antibodies to M13 procoat will precipitate procoat but not coat, suggesting that the antibody molecules are specifically recognizing the leader sequence or the conformation which it induces in the whole procoat molecule.

Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences.

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.

Purification and characterization of leader (signal) peptidase from Escherichia coli.

Many membrane proteins and secreted proteins are synthesized in precursor form with 15 to 30 additional NH2-terminal residues. These "leader peptides" (pre-pieces, signal peptides) are removed as these proteins cross or insert into cellular membranes. "Leader peptidase" activities which catalyze this cleavage have been detected in crude extracts and found to be dependent on membrane fractions. We now describe a 6,000-fold purification of a leader peptidase from the membranes of uninfected Escherichia coli. This leader peptidase was assayed by its ability to cleave the 23-residue leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Immunoprecipitation and amino acid sequencing showed that this enzyme cleaved procoat to produce authentic coat protein. No factors other than the leader peptidase were found to be required for the conversion of procoat protein to coat protein.

Proteinaceous receptors for the import of mitochondrial precursor proteins.

Mild trypsin treatment of isolated Neurospora mitochondria strongly inhibits their ability to bind and import the precursors of several mitochondrial proteins. Evidence is presented for two proteins, the ADP/ATP carrier and the mitochondrial porin, that specific binding of the precursors to the outer surface of the mitochondria is affected by the protease treatment. We suggest that the receptors that mediate the import of these two precursors are proteinaceous. Treatment of mitochondria with elastase also inhibits the binding and import of the ADP/ATP carrier and the porin. In contrast the import of the precursors of subunits 2 and 9 of the mitochondrial proton-translocating ATPase was unaffected by elastase treatment at the concentrations used. We suggest that the import pathways of the latter two proteins are distinct from those of the ADP/ATP carrier and the porin.

Peroxidative damage to cardiac mitochondria: identification and purification of modified adenine nucleotide translocase.

Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118-123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu2+/t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.

Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria.

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.

Leader peptidase is found in both the inner and outer membranes of Escherichia coli.

Many membrane proteins are synthesized as transient precursors with an NH2-terminal leader (or signal) peptide. During insertion of these proteins into the membrane, leader peptides are removed by leader peptidase. One such enzyme has been detected in detergent extracts of Escherichia coli membranes and extensively purified using as an assay the removal of the leader sequence of procoat, the precursor of the major coat protein of bacteriophage M13. We now report that this leader peptidase is found in equal abundance in the inner and outer membranes of E. coli. Enzyme from each membrane accurately cleaves procoat to mature M13 coat protein. The salt, pH, and Mg2+ optima and inhibitor sensitivities of enzyme from each membrane are identical. Furthermore, the activities are indistinguishable upon ion exchange chromatography and nondenaturing gel electrophoresis. Finally, a strain of E. coli with a plasmid which causes overproduction of leader peptidase has elevated levels of enzyme in both the inner and outer membranes. Leader peptidase is the only known enzyme which is found in both inner and outer membrane fractions of E. coli; this may reflect its role in membrane biogenesis.

A rapid method for the preparation of yeast for immunoelectron microscopy using Lowicryl HM-20.

We describe a fixation and embedding procedure for the yeast Saccharomyces cerevisiae using Lowicryl HM-20 which is rapid, gives excellent fixation, and avoids the low temperature handling normally associated with embedding in this resin. This procedure yields superior structural preservation when compared to the commonly used rapid embedding procedure which employs Lowicryl K4M. We demonstrate that sections prepared using our rapid procedure are suitable for use in immunogold labelling experiments.

Free radical-induced alterations of myocardial membrane proteins.

Rat myocardial membranes exposed to the free radical-generating systems, Fe2+/ascorbate, Cu2+/t-butylhydro-peroxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The "28K reaction" occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.

Peroxidative damage to cardiac mitochondria. II. Immunological analysis of modified adenine nucleotide translocase.

We have previously reported that treatment of isolated rat heart mitochondria with the free radical-generating system Cu2+/tert-butylhydroperoxide produces striking changes in the adenine nucleotide translocase (ANT) of the inner membrane. These changes include a small increase in apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by its disappearance from the polypeptide profile upon further oxidant treatment (Zwizinski and Schmid (1992) Arch. Biochem. Biophys. 294, 178-183). In order to characterize its peroxidative modification in more detail, we have purified rat heart ANT and prepared polyclonal antibody against it. Using this antibody, we have observed that increasing oxidant treatment results in a gradual increase in the ANT protein's apparent molecular weight by up to 1 kDa. The progressive nature of the molecular weight shift, which parallels the generation of thiobarbituric acid reactive substances, supports the hypothesis that this phenomenon may be the result of covalent addition of increasing amounts of lipid peroxidation products. Strong oxidative treatment of cardiac mitochondria also causes fragmentation and polymerization of the ANT protein. However, Western blot analysis showed that a major portion of the original ANT survives even extensive oxidation as a distinct, modified protein. Therefore, the almost complete disappearance of ANT from Coomassie-stained gels appears to be the result of cross-linking and fragmentation reactions, as well as a decreased efficiency of the Coomassie staining. Because a measurable molecular weight shift of ANT occurs at the mildest oxidative treatment tested (resulting in the production of only 1.1 nmol malondialdehyde/mg protein), it may be relevant as a parameter of myocardial ischemia-reperfusion injury.

Release of mitochondrial matrix proteins through a Ca2+-requiring, cyclosporin-sensitive pathway.

Induction of the inner membrane permeability transition, normally associated with the release of small molecules and ions from the mitochondrial matrix, also causes the release of matrix proteins. The release is linear with time and slow when compared to the time course of mitochondrial swelling. Transient induction of the high permeability state is reflected in transient release of proteins. Cyclosporin A (0.5 nmol/mg protein) or chelation of free Ca2+, which reverses the permeability transition, also block the subsequent release of protein even when added after extended preincubation. Possible mechanisms of protein release are discussed.

Mechanisms of membrane assembly: effects of energy poisons on the conversion of soluble M13 coliphage procoat to membrane-bound coat protein.

The coat protein (gene 8 product) of coliphage M13 spans the host cell plasma membrane prior to its assembly into extruding virions. It is made as a soluble precursor, termed procoat, with an extra 23 NH2-terminal amino acid residues. We have examined the effect of metabolic poisons on the assembly of procoat into the plasma membrane and its proteolytic conversion to coat protein. Protein synthesis and proline uptake were measured to assess the effect of each poison on cellular high-energy phosphate and on the transmembrane protonmotive force, respectively. Arsenate, which abolished protein synthesis but did not affect proline uptake, had no measurable effect on the conversion of procoat to coat protein. In contrast, the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) blocked conversion of procoat to coat protein. Neither CCCP nor arsenate inhibited the ability of a detergent-solubilized and highly purified preparation of leader peptidase to convert procoat to coat protein in the presence of detergents. The procoat that accumulated in the presence of CCCP was membrane bound. A spontaneous mutant that grows in the presence of CCCP showed (i) CCCP-resistant proline uptake in whole cells, (ii) CCCP-resistant uptake in inner membrane vesicles, and (iii) CCCP-resistant conversion of procoat protein to coat protein. These data suggest that an electrochemical gradient is at least indirectly necessary for the proper assembly of procoat into the cellular membrane.

Synthesis of phage M13 coat protein and its assembly into membranes in vitro.

The coat protein (gene 8 product) of coliphage M1O is an integral protein of the host cell membrane at all stages of virus infection. This protein, when made in a cell-free reaction, has been shown by others to have an additional NH2-terminal peptide region and is referred to as "procoat." It is initially not membrane-bound but, upon exposure to Escherichia coli membrane vesicles or to liposomes prepared from E. coli lipids, it assembles into the bilayer in an integral fashion. Much of this protein is shown to be exposed on the inner surface of the liposome. We suggest that refolding of procoat as it encounters the bilayer is sufficient to transport large segments of the peptide chain through the apolar hydrocarbon core.