Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.     

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.     

Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells

Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

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.     

mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, we show that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way, by which the oocyte implements polarized deposition of transmembrane/secreted proteins. We propose that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons.     

Fertilization in a Crab. III. Cytodifferentiation of vesicles enclosing ring-shaped elements involved in the cortical reaction

During the cortical reaction, Carcinus maenas eggs successively released a fine granular material and a massive amount of ring-shaped elements that subsequently formed most of the fertilization envelope. The ring-shaped elements came from egg cortical vesicles and, owing to their striking morphology, acted as naturally occurring markers in ultrathin sections, which permitted us to understand the pathway of their intracellular transport. In this respect it was established that the ring-shaped elements and their enclosing vesicles originated in the endoplasmic reticulum both in ripe oocytes and early fertilized eggs maintained under in vitro conditions. The intracellular transport pathway of the endoplasmic reticulum-derived vesicles seemed to bypass the Golgi apparatus. Accordingly, the ring-shaped elements appeared to be released by direct exocytosis from the endoplasmic reticulum system. Finally, a tentative scheme of oocytes functioning is suggested for crustacean decapods, based on the remarkable similarities between the structure and ER origin of the ring-shaped elements involved in the cortical reaction and the disc-shaped granules traditionally considered as endogenous yolk precursors. The scheme implies that the oocyte ER system might produce a precursor common to the cortical reaction exudate and to the endogenous yolk, in the form of the ring- or disc-shaped elements.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).     

Transport between ER and Golgi

In the last eighteen months, it has become clear that some classes of proteins are actively recruited into endoplasmic reticulum export carriers, whereas others are exported as bulk-flow cargo. Subsequent transport to the Golgi is mediated by tubulovesicular membranes. The anterograde membrane flow is compensated for by a retrograde pathway, which, in addition to the recycling of membrane and proteins to the endoplasmic reticulum, may play a role in anterograde cargo concentration.     

Reproductive biology of the polychaete Kefersteinia cirrata Keferstein (Hesionidae). I. Ovary structure and oogenesis

The ultrastructure of the ovary and the developing oocytes of the polychaete Kefersteinia cirrata have been described. The paired ovaries occur in all segments from the 11th to the posterior. Each consists of several finger-like lobes around an axial genital blood vessel. Oogenesis is well synchronised, young oocytes start to develop in September and vitellogenesis begins in January and is completed by May.

The young oocytes are embedded among the peritoneal cells of the blood vessel wall which have accumulations of glycogen and other storage products. Each oocyte becomes associated with a follicle cell with abundant rough endoplasmic reticulum. Yolk synthesis involves the accumulation of electron dense granules along the cisternae of the abundant rough endoplasmic reticulum. Active Golgi complexes are present and are involved in the production of cortical alveoli. The oocyte has branched microvilli, which contact the follicle cells or blood sinuses between the follicle cells and peritoneal cells. In post-spawning individuals the lysosome system of the follicle cells is hypertrophied and the cells play a role in oocyte breakdown and resorption.     

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.     

Fibroblast growth factor 9 secretion is mediated by a non-cleaved amino-terminal signal sequence

Fibroblast growth factors are a family of intercellular signaling molecules with multiple and varied roles in animal development. Most are exported from cells by means of a classical amino-terminal signal sequence that is cleaved from the mature protein during its passage through the secretory pathway. Fibroblast growth factor-9 (Fgf-9) does not contain a recognizable signal sequence, although it is efficiently secreted. In this study, we show that Fgf-9 enters the endoplasmic reticulum and traverses the Golgi complex in a similar manner to other constitutively secreted proteins. Deletion and point mutation analysis has revealed an atypical non-cleaved signal sequence within the amino-terminal region of Fgf-9. Moreover, the first 28 amino acids of Fgf-9 can function as an efficient non-cleaved signal peptide when appended to the amino terminus of green fluorescent protein.     

Alpha 1-antitrypsin deficiency—A defect in secretion

A naturally occurring point mutation in the human alpha 1-antitrypsin gene leads to the synthesis of a variant of the protein which is poorly secreted from hepatocytes. This Z; mutation codes for a glutamic acid to lysine substitution at residue 342 in the polypeptide chain. The mutant protein is correctly translocated into the lumen of the endoplasmic reticulum and core glycosylated but inefficiently transported beyond the ER compartment. Experiments using Xenopus oocytes as a surrogate secretory cell show that abberant secretion of the variant is not confined to hepatocytes and glycosylation of the polypeptide is not obligatory for the block in secretion. Site-directed mutagenesis can be used to examine the effect of natural mutations on protein structure and the relationship between structure and intraceltular transport.     

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.     

Degradation of proteins within the endoplasmic reticulum.

Certain newly synthesized proteins within the endoplasmic reticulum undergo rapid turnover by a non-lysosomal proteolytic pathway. Biochemical and morphological evidence has suggested that these proteins never leave the endoplasmic reticulum before they are degraded. The mechanism(s) for the selective targeting of proteins for degradation within the endoplasmic reticulum is still not understood, but appears to rely on specific structural determinants on the protein substrates. Important cellular functions are likely to be served by this endoplasmic reticulum degradative system, including disposal of abnormal proteins and the selective turnover of metabolically regulated proteins.     

The N- and C-terminal regions regulate the transport of wheat gamma-gliadin through the endoplasmic reticulum in Xenopus oocytes.

Following sequestration into the endoplasmic reticulum (ER), wheat storage proteins are naturally either retained and packaged into protein bodies within this organelle or exported to the Golgi apparatus. To identify protein domains that control the sorting of wheat storage proteins within the ER, a wild-type gamma-gliadin storage protein as well as two of its deletion mutants, each bearing either of the two autonomous N- and C-terminal regions, were expressed in Xenopus oocytes. Our results demonstrated that the N-terminal region of the gliadin, which is composed of several tandem repeats of the consensus sequence PQQPFPQ, was entirely retained within the ER and accumulated in dense protein bodies. In contrast, the C-terminal autonomous region was efficiently secreted to the medium. The wild-type gamma-gliadin, containing both regions, was secreted at a lower rate and less efficiently than its C-terminal region. These results suggest that sorting of the wheat gamma-gliadin within the ER may be determined by a balance between two opposing signals: one functions in the retention and packaging of the storage protein within the ER, while the second renders the protein competent for export from this organelle to the Golgi apparatus.     

Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin

The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.     

Cytosolic and nuclear aggregation of the amyloid beta-peptide following its expression in the endoplasmic reticulum

Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid beta-peptide (Abeta) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Abeta aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Abeta forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.     

FAD oxidizes the ERO1-PDI electron transfer chain: the role of membrane integrity

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.