Synthesis and insertion, both in vivo and in vitro, of rat-liver cytochrome P-450 and epoxide hydratase into Xenopus laevis membranes

We described whole cell and cell-free systems capable of inserting into membranes cytochrome P-450 and epoxide hydratase made under the direction of rat liver RNA. The systems have been used to study the pathways followed by newly made secretory and integral membrane proteins. The cell-free system contains Xenopus laevis embryo membranes, and demonstrates competition for a common receptor between cytochrome P-450 and epoxide hydratase, and normal secretory proteins: evidence is provided for differential membrane receptor affinity. Thus, synthesis of secretory and membrane proteins appears to involve a common initial pathway. Microinjection of rat liver RNA into whole oocytes suggests that membrane insertion is neither cell type nor species specific, because functional rat liver enzymes are found inserted in the endoplasmic reticulum of the frog cell. Nonetheless, insertion is highly selective since albumin and several other proteins made under the direction of the injected liver RNA are sequestered within membrane vesicles and are then secreted by the oocyte, whilst epoxide hydratase and cytochrome P-450 are inserted into membranes but are not secreted.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Decay of the oocyte-type heat shock response of Xenopus laevis

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.     

The influence of topology and glycosylation on the fate of heterologous secretory proteins made in Xenopus oocytes

Secretory proteins made in Xenopus laevis oocytes under the direction of heterologous messenger RNA are modified, topologically segregated and exported. Thus the oocyte may serve as a useful surrogate secretory system and we have studied some of the factors governing access to the export pathway. Unglycosylated chicken ovalbumin, synthesized and trapped in the cytosol, is not secreted but glycosylated ovalbumin, found sequestered within vesicles, is exported from oocytes. However, ovalbumin, which is transferred across the endoplasmic reticulum in the presence of tunicamycin and which is indistinguishable by immunoprecipitation, by two-dimensional gel electrophoresis and by concanavalin-A--Sepharose binding from the cytosolic form, is still secreted. Guinea-pig milk proteins and human interferon are also exported from tunicamycin-treated frog cells. These observations demonstrate that access to the endoplasmic reticulum but not glycosylation is a mandatory intermediate step in secretion, and emphasize the advantages of the oocyte as a surrogate system for the study of the later events in the gene expression pathway.     

Physiology of rat-liver polysomes: Protein synthesis by stable polysomes

Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Effect of a short term fast on the distribution of cytoplasmic albumin messenger ribonucleic acid in rat liver. Evidence for formation of free albumin messenger ribonucleoprotein particles

Recently, using molecular hybridization techniques with albumin [3H]cDNA, we have determined that in normally fed rats 98% of total liver polyribosomal albumin mRNA sequences are found in membrane-bound polyribosomes (Yap, S. H., Strair, R. K., and Shafritz, D. A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5397-5401). We now observe that a 24- to 30-h withdrawal of food leads to major changes in the amount and subcellular distribution of albumin mRNA molecules. The total amount of cytoplasmic albumin mRNA per liver and concentration of albumin mRNA per unit of membrane-bound polyribosomal RNA are decreased. However, the proportion of albumin mRNA present in the postribosomal supernatant fraction increases dramatically in a short term fast, so that it now represent 60% of total cytoplasmic albumin mRNA sequences. Most of the albumin mRNA sequences in the postribosomal supernatant fraction sediment between 30 S and 50 S. These findings suggest that albumin mRNA is probably stored in the messenger ribonucleoprotein fraction during the fasting state.     

Distribution of mRNAS of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of alpha-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.     

Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.     

A Freeze-Sectioning Method for Preparation of the Detergent-Resistant Cytoskeleton Identifies Stage-Specific Cytoskeleal Proteins and Associated mRNA in Xenopus Oocytes and Embryos

Proteins of the detergent-resistant cytoskeleton fraction and the detergent-soluble fraction from Xenopus oocytes and embryos are examined using a procedure which allows rapid and uniform extraction of tissues and large, single cells. SDS-polyacrylamide gels reveal only a few prominent cytoskeletal proteins in the early embryo, however qualitatively different proteins begin to appear after gastrulation. Incorporation of [³⁵S]-methionine into newly synthesized proteins indicates that there is synthesis and assembly of proteins into the cytoskeleton, but the amount remains low until after gastrulation. The use of nucleic acid probes for alpha-tubulin and actin mRNA indicates that about 80% of these mRNAs in the oocyte and meiotically mature egg are bound to the detergent-resistant cytoskeleton.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.