Mutant yolk proteins lead to female sterility in Drosophila.

Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.     

Yolk polypeptide secretion and vitelline membrane deposition in a female sterile Drosophila mutant

Ovarian follicle cells of wild type Drosophila melanogaster simultaneously secrete yolk polypeptides (YP1, YP2 and YP3) and vitelline membrane proteins. In order to understand the relationship between these two secretory activities, we have investigated the ultrastructure of a female sterile mutation that alters YP1 secretion and vitelline membrane deposition. Homozygous fs(1)1163 females lay eggs that collapse and contain reduced quantities of YP1. Secretory granules in follicle cells contain an electron-translucent component that is assembled into the developing vitelline membrane in both mutant and wild-type ovaries, and an electron-dense component that disperses after secretion in wild-type ovaries. Mutant ovaries differ from wild-type by (1) having larger secretory granules (2) forming clumps of the dense secretory component within the developing vitelline membrane (3) accumulating more tubules in the cortical ooplasm of vitellogenic oocytes, and (4) possessing altered yolk spheres. Mutant ovaries implanted into wild-type hosts showed no improvement in the secretory granules and slight improvement in the vitelline membrane clumps but amelioration of the oocyte phenotypes. Since genetic evidence suggests that the fs(1)1163 mutation resides in or near the Yp1 gene and biochemical data show that the mutation alters YP1 structure, we conclude that the ultrastructural phenotypes are due to a structurally abnormal YP1 in the mutant. The alteration in vitelline membrane structure caused by the dense clumps could account for collapsed eggs and, hence, the female sterility of the mutant.     

A yolk protein mutant leads to defects in the secretion machinery of Drosophila melanogaster.

The three yolk proteins of Drosophila melanogaster are synthesized in the fat body and ovarian follicle cells. A mutation in yolk protein 3, YP3S1, has been described in which the leader sequence is not cleaved from the protein. We describe here ultrastructural and molecular studies on the YP3S1 mutant and show that the mutant protein enters the secretory pathway and forms precipitates, often as electron dense material in excessive elaborations of the plasma membrane. Females homozygous for YP3S1 lay fewer eggs than wild type flies and these embryos are less viable. The abnormal ultrastructure of the yolk spheres observed suggests that whilst YP3 is not completely essential for viability, it is required for normal yolk sphere morphogenesis.     

Sequence analysis of a yolk protein secretion mutant of Drosophila melanogaster

Summary The female-sterile mutants fs(1) 1163 of Drosophila melanogaster described by Gans et al. (1975) has been characterised as a yolk protein 1 (YP1) secretion mutant (Bownes and Hames 1978b; Bownes and Hodson 1980). We have cloned and sequenced the YP1 gene from this strain, and the strain in which the mutant was induced. One amino acid substitution was found in the predicted polypeptide sequence, an isoleucine to asparagine change at position 92. The sequence of the leader peptide was identical to previously published YP1 sequences. The possible effects of the amino acid change were investigated by computer analysis, which suggests there is no major alteration of secondary structure, but that a hydrophobic region in YP1 is lost in the mutant. This may affect higher order structure.     

Processing and secretion of a mutant yolk polypeptide in Drosophila

Flies homozygous for the female sterile mutation fs(1)1163 produce eggs deficient in YP1, one of the three major yolk polypeptides. Genetic studies showed that fs(1)1163 is cis acting on YP1 quantity, so that mutation does not control a diffusible substance regulating YP1 production. The sterility and YP1 quantity phenotypes were not genetically separated from each other or from the structural gene for YP1, indicating that the mutation is located in or near Yp1. The amount of translatable YP1 message in mutant and wild-type cells was approximately equal, but the primary translation products were different in size and, hence, different in structure. The signal peptide was cleaved normally from the mutant polypeptide, and phosphorylation and glycosylation of the mutant YP1 also occur. However, YP1 processing intermediates that are transient in wild-type cells become major species in fs(1)1163 cells. We conclude that fs(1)1163 alters the primary structure of YP1 in a way that does not block signal-peptide cleavage but does alter later processing steps and hence its rate of secretion, leading to the YP1 deficiency found in the hemolymph and eggs.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.     

Mutant fs(1) 1163 of Drosophila melanogaster alters yolk protein secretion from the fat body

Summary The mutant fs(1) 1163 of Drosophila melanogaster, which was isolated by Gans et al. (1975) is a recessive homozygous female sterile at 18°C and a dominant female — sterile at 29°C. We reported previously that there are reduced quantities of the largest of the three yolk polypeptides in Drosophila melanogaster in the haemolymph and eggs of this mutant at 29°C (Bownes and Hames 1978 a). In this paper we show that the yolk protein defect maps within approximately 2.5 recombination units of the female sterility at 21±2.5 map units on the X-chromosome. The temperature-sensitive period of the yolk protein defect is after emergence. In vitro labelling of fs(1) 1163 ovaries and fat bodies showed that they were able to synthesise yolk polypeptide 1. Interestingly, studies on the proteins present in the various tissues indicate that the fat body tends to accumulate all three yolk polypeptides in the mutant. This phenotype is partially co-dominant in that an effect is seen in heterozygotes as well as homozygotes and is enhanced by increased temperature. This mutant could therefore have a defect (a) in the structural gene for yolk polypeptide 1, (b) in the processing and secretion enzyme systems; (c) in the fat body or all tissues leading to altered secretion properties.Mutants like fs(1) 1163 which alter specific steps in vitellogenesis should be of value for analysing the genetic and biochemical control of the synthesis, transport and sequestering of the yolk polypeptides during oogenesis.     

Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes

The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP). Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae. RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells. Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes. Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP. Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed. Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development. These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes. This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.     

Comparison of Yolk Production in Seven Pyralid Moth Species

Summary

The yolk proteins of six pyralid moths were analyzed and compared with the yolk proteins of Plodia interpunctella (Hübner). When cross-reacted in an Ouchterlony double immunodiffusion with antiserum raised to either total yolk proteins or purified vitellin from P. tnterpunctella, the yolk proteins of Anagasta kuehniella (Zeller), Cadra cautella (Walker), C. figulilella (Gregson), and Ephestia elutella (Hübner), closely related members of the subfamily Phycitinae, showed strong precipitation lines that consisted of four major yolk polypeptides (YPs). The yolk proteins from Amyelois transitella (Walker) were only weakly reactive, whereas yolk proteins from Galleria mel-lonella (L.) were not precipitated by either antiserum. Abdominal body walls (containing primarily fat body) from late pharate adult females were incubated in vitro and they secreted two major polypeptides that had molecular masses similar to the vitellogenins (YP1 and YP3) from P. interpunctella. In addition, ovarioles from late pharate adult females were incubated in vitro, and they secreted two major polypeptides that had molecular masses similar to YP2 and YP4 from P. interpunctella. When late pharate adult females were injected with ³⁵S-Met, the hemolymph of all species contained vitellogins that were secreted by their respective body walls in vitro. Ovarioles from injected females contained many labeled polypeptides, but there were four major bands that corresponded consistently to the vitellogenins secreted from the fat body and the two major polypeptides secreted from the ovarioles. These data show that the production of the major YPs in these closely related pyralid species is very similar, and that there is considerable conservation of immunological characters of yolk proteins in the subfamily Phycitinae.     

Identification of a female-sterile mutation affecting yolk protein 2 in Drosophila melanogaster

Summary The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperaturesensitive female-sterile mutant, fs(1) K313, and characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype.     

Use of promoter fusions in Drosophila genetics: characterization of a YP1-ADH fusion gene

In Drosophila melanogaster the yolk protein (YP) genes are normally expressed only in the fat body and follicular epithelium of adult females--never in males or in larvae. We describe here a first step toward a genetic examination of the developmental controls that restrict the activity of the YP genes to adult female tissues. A YP1 promoter that contains the tissue-, temporal-, and sex-specific controlling elements for expression was fused to the reporter gene, alcohol dehydrogenase (Adh). The gene fusion was transformed into an Adh-deficient genotype. As assayed by a number of criteria, that the fusion gene is expressed in the same physiological manner as the endogenous yolk protein genes. The fusion gene's activity is modulated in trans by a temperature-sensitive allele of the sex determination gene, tra-2. The Adh enzyme serves as a selectable marker and therefore these flies are suitable for use in genetic screens for trans-acting mutations that affect the expression of the yolk protein genes.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Biosynthesis of Drosophila yolk polypeptides

The three yolk polypeptides (YPs) of Drosophila are synthesized and secreted by female fat body and ovarian follicle cells, sequestered by pinocytosis into oocytes, and finally deposited into yolk granules. The biosynthesis of the YPs was studied using two-dimensional gels. Labeling the YPs with [³⁵S]-cysteine, an amino acid found only near the amino terminus of YP1 and YP2, showed that an amino terminal peptide is removed from YP1 and YP2 shortly after or during translation. Intermediates in YP biosynthesis corresponding in electrophoretic mobility to pancreatic membrane-processed primary translation products were also detected in a 5-min pulse label with [³⁵S]-methionine. Genetic variants that alter YP structure were used to identify which YP precursor comes from which Yp gene. Pulse labeling with [³⁵S]-methionine revealed that all three YPs becomes more negatively charged, that YP1 and YP2 become heterogeneously charged, and that YP1 gains in apparent molecular weight within 15 min after translation. Injecting female flies with radioiabeled sugars or orthophosphate revealed that the YPs are glycosylated and phosphorylated. Treating hemolymph proteins with phosphatase showed that phosphorylation is responsible for much of the change in charge and increase in molecular weight of the maturing YPs. These experiments with wild-type flies provide a basis for the analysis of mutations at the Yp genes which alter the structure of individual YPs.     

Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with ³⁵S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsx^D, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.     

Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.     

CDNA cloning and characterization of S6 kinase and its effect on yolk protein gene expression in the oriental fruit fly Bactrocera dorsalis (Hendel)

p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of target of rapamycin (TOR) gene and an important regulator of protein synthesis responsible for cell growth and reproduction. In this study, a S6K gene, named BdS6K (GenBank Accession No. GQ203802), was isolated from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed that BdS6K mRNA is expressed at a higher level in egg than in other developmental stages, as well as in ovary than in fat body. Downregulation of BdS6K activity by rapamycin treatment in larval stage resulted in the developmental defects of larvae, pupae, and adults, with a reduced yolk protein (YP) expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size, and also repressed the egg development in female fruit fly. Knockdown of BdS6K gene by RNA interference in the adult significantly decreased the YP expression. These observations support the involvement of BdS6K signaling in the regulation of the YP synthesis and egg development in B. dorsalis.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.