Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria.

Treatment of isolated yeast mitochondria with high levels (1 mg/ml) of trypsin severely inhibits protein import but does not destroy the integrity of the outer membrane or abolish mitochondrial energy coupling. If the outer membrane of these trypsin-inactivated mitochondria is disrupted by osmotic shock, the resulting mitoplasts are again able to import proteins. Protein import into mitoplasts, like that into intact mitochondria, is energy-dependent; however, whereas import into mitochondria is inhibited by antibody against 45-kd proteins of the outer membrane [Ohba and Schatz, EMBO J., 6, 2109-2115 (1987)], import into mitoplasts not affected by this antibody. Protein import into mitoplasts appears to bypass one or more steps normally occurring at the mitochondrial surface.     

Dissociation of yeast 80 S ribosomes by chaotropic salts

Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO₄ or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO₄ was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed.     

Attachment of cells to basement membrane collagen type IV

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.     

Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.     

Cytoplasmic p53 polypeptide is associated with ribosomes.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.     

Structure of the yeast ribosomes. Proteins associated with the rRNA.

Polyamines have been shown to bind to doubled stranded regions of rRNA [3]. Therefore, ribosomal proteins that can be cross linked to these molecules in the ribosomes structure must be bound to or located in the vicinity of the RNA. This technique is the first to yield results on the proteins associated with the rRNA in the eukaryotic ribosome where the lack of purified ribosomal proteins does not allow the use of direct binding studies as in bacterial systems. Proteins S7, S10, S13, S21, S22 and S27 in the small subunit and L2/3, L5, L10/12, L19/20, L22, L23, L36/37, L42 and L43' in the large subunit are labelled when cross linked to [14C]spermidine using 1,5-difluoro 2,4-dinitrobenzene and are good candidates to be RNA-binding proteins in ribosomes from Saccharomyces cerevisiae.     

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.     

The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles

The yeast mitochondrial outer membrane was isolated and 10 of its major polypeptides were identified (mol. wts. 109, 70, 57, 45, 45, 42, 33, 29, 25 and 14 kd). The membrane has no major polypeptide in common with either mitochondrial inner membrane or rough microsomes. Protease treatment and immunochemical techniques showed that virtually all of the isolated outer membrane vesicles are sealed and display the same surface orientation as in the intact mitochondrion.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Attachment of muscle filaments to the outer membrane of the nuclear envelope

Summary Myofilaments of striated muscles can be recognized in the electron microscope to be in structural continuity with the outer membrane of the nuclear envelope. The very site of insertion of these myofilaments at the membrane surface frequently appears characterized by a dense basal knob of 85–135 Å. It is hypothesized that this attachment of myofilaments to the nuclear membrane plays a role in mechanically transmitting the contraction of the fiber to the nucleus, thus bringing about the harmonica-like folded appearance of the nucleus which is known for the contracted states of striated, smooth and cardiac muscles.The work was supported in part by the Deutsche Forschungsgemeinschaft.The author is indebted to Miss Sigrid Krien and Miss Marianne Winter for careful technical assistance as well as to Drs. Heinz Falk and U. Scheer for valuable discussions.     

The release and reassociation of 5.8 S rRNA with yeast ribosomes.

Yeast 5.8 S rRNA is released from purified 26 S rRNA when it is dissolved in water or low salt buffer (50 mM KCl, 10mM Tris-HCl, pH 7.5); it is not released from 60 S ribosomal subunits under similar conditions. The 5.8 S RNA component together with 5 S rRNA can be released from subunits or whole ribosomes by brief heat treatment or in 50% formamide; the Tm for the heat dissociation of 5.8 S RNA is 47 degrees C. This Tm is only slightly lower when 5 S rRNA is released first with EDTA treatment prior to heat treatment. No ribosomal proteins are released by the brief heat treatment. A significant portion of the 5.8 S RNA reassociates with the 60 S subunit when suspended in a higher salt buffer (e.g.0.4 m KCl, 25 mM Tris-HCl, pH 7.5, 6 mM magnesium acetate, 5 mM beta-mercaptoethanol). The Tm of this reassociated complex is also 47 degrees C. The results indicate that in yeast ribosomes the 5.8 S-26 S rRNA interaction is stabilized by ribosomal proteins but that the association is sufficiently loose to permit a reversible dissociation of the 5.8 S rRNA molecule.