Functional interaction of plant ribosomes with animal microsomal membranes.

Translation of mRNA for the light chain of murine immunoglobulin in a wheat germ cell-free system in the presence of stripped microsomal membranes from canine pancreas resulted in co-translational proteolytic conversion of the precursor of the light chain, reducing it to the size of the authentic light chain of immunoglobulin, and in co-translational segregation of the processed chains in a proteolysis resistant space of the heterologous microsomal vesicles.     

Identification of rabbit microsomal cytochrome P-450 isozyme,form 1, as a hepatic progesterone 21-hydroxylase

Six highly purified forms of rabbit microsomal cytochrome P-450, isolated from hepatic microsomes, exhibit differences in the regiospecific metabolism of progesterone. Only one of the isozymes studied, form 1, catalyzes the formation of deoxycorticosterone from progesterone at an appreciable rate. This cytochrome P-450 isozyme may participate in the conversion of progesterone to deoxycorticosterone during pregnancy. All six forms of cytochrome P-450 catalyze 6β- and 16α-hydroxylation at the two concentrations of progesterone tested. Form 3b exhibits a lower apparent Km for 6β-hydroxylation than the other five.     

Identification of an Escherichia coli inner membrane polypeptide specified by a lambda-tonB transducing.

Analysis by polyacrylamide gel electrophoresis of the proteins coded by a λ$\text{ton}$B transducing phage, after infection of UV-irradiated bacteria, revealed the presence of at least 7 new polypeptides. Three of these were identified as proteins of the $\text{trp}$ operon whilst three others were deleted by a spontaneous mutation in the $\text{ton}$B region carried by the phage. A single polypeptide, molecular weight 40,000 was absent from a phage carrying a proflavine induced mutation in $\text{ton}$B. We conclude that this protein, which was localised in the inner membrane by sarkosyl fractionation of the envelope, is the $\text{tonB}$ product.     

Distant segments of Saccharomyces cerevisiae scR1 RNA promote assembly and function of the signal recognition particle

The conserved signal recognition particle targets ribosomes synthesizing presecretory proteins to the endoplasmic reticulum membrane. Key to the activity of SRP is its ability to bind the ribosome at distant locations, the signal sequence exit and elongation factor-binding sites. These contacts are made by the S and Alu domains of SRP, respectively. We tested earlier secondary structure predictions of the Saccharomyces cerevisiae SRP RNA, scR1, and provide and test a consensus structure. The structure contains four non-conserved insertions, helices 9-12, into the core SRP RNA fold, and an extended helix 7. Using a series of scR1 mutants lacking part or all of these structural elements, we find that they are important for the RNA in both function and assembly of the RNP. About 20% of the RNA, corresponding to the outer regions of these helices, is dispensable for function. Further, we examined the role of several features within the S-domain section of the core, helix 5, and find that its length and flexibility are important for proper SRP function and become essential in the absence of helix 10, 11 and/or 7 regions. Overall, the genetic data indicate that regions of scR1 distant in both primary sequence and secondary structure have interrelated roles in the function of the complex, and possibly mediate communication between Alu and S domains during targeting.     

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane

Hybridization-selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell-free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium-washed membranes (K-RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K-RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557-561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.     

Insulin stimulates phosphorylation of a heat-stable protein in rat adipose tissue

Insulin in rat adipose tissue acts to increase the phosphorylation about 2.5-fold of a low molecular weight protein in the cytosol designated phosphoprotein m. Isoproterenol had no effect on the phosphorylation of phosphoprotein m. Some of the properties of phosphoprotein m are: soluble in 1% trichloro acetic acid, heat-stable and has a molecular weight of 23,000 on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphoserine and phosphothreonine are the phosphorylated amino acid residues of phosphoprotein m. The physical and chemical properties of phosphoprotein m are similar to those of previously described inhibitor and modulator proteins.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

Induced changes in the affinity of 1,25-dihydroxyvitamin D3 receptors in chick intestine

The contents of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in plasma and intestinal mucose were increased by dietary calcium and by dietary phosphorus restriction. The concentration of intestinal occupied receptors for 1,25(OH)2D3 was higher in calcium-restricted birds. The affinity (association constant) of intestinal receptors for 1,25(OH)2D3 was lower in phosphorus-restricted chicks, as compared to control or calcium-restricted chicks. The number of binding sites were not influenced by dietary calcium or phosphorus restriction.     

Susceptibility of platelet alpha-actinin to a Ca2+-activated neutral protease

Purified alpha-actinin from human platelets was digested with Ca2+-activated protease from muscle. The alpha subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, alpha-actinin is cleaved by the endogenous Ca2+-activated protease.     

Passage of viral membrane proteins through the Golgi complex

BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes.     

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6–7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b₅ content, NADH and NADPH cytochrome c reductase activities (1.0–1.5-fold). Only very small changes in the total content of cytochrome P-450 were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of M_r = 48 000; the faster-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67 000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.     

Purification and partial characterization of a 19kD/pI 4.5 nucleolar phosphoprotein

Two-dimensional PAGE analysis of proteins associated with the slowly sedimenting "fibrillar" structures of HeLa nucleoli revealed a protein with a M of 19,000 and a pI of 4.5 which was highly labeled both with 32P-orthophosphate and 35S-methionine. The protein was isolated from Novikoff hepatoma nucleoli by extraction in 0.35 M NaCl and 5 mM DTT followed by chromatography in EDTA on DEAE-cellulose and Sephadex G-100. The protein was homogeneous with respect to two-dimensional PAGE, number of tryptic peptides and carboxyl terminal analysis. The protein contained an acidic/basic amino acid ratio of 2.1, 7 residues of methionine, 2 residues of cysteine, a blocked amino terminus and a carboxyl terminal lysylleucine.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeant labeling experiments indicated that approximately 40% of these proteins were exposed on the extracytoplasmic surface of the plasma membranes of these cells. The studies demostrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.