Interlayer Trafficking of Drosophila Eggshell Proteins

Waring, Gail

Abstract

The long term goals of this work are to understand the morphogenesis of the eggshell in Drosophila, to contribute to the understanding of the role of the extracellular matrix surrounding the oocyte in specifying the major axes of the egg and to extend the understanding of how complex extracellular architectures are constructed. The specific goal is to understand the requirements for proper deposition of two of at least five products of the defective chorion (dec-1) gene in Drosophila melanogaster. The dec-1 gene is required to organize the chorion proteins into a tripartite structure and in dec-1- homozygotes, the chorionic material collapses into the vitelline membrane by late stage 14. Considerable preliminary work has already been done. The dec-1 gene produces three alternatively spliced RNAs whose protein products are subjected to limited proteolysis to create a variety of proteins with distinctive 3' and 5' ends. The most abundant of these is fc-106. Using antibodies raised against different portions of this proprotein, Dr. Waring has demonstrated that it is cleaved into an N-terminal 25 kD and a C-terminal 80 kD polypeptide and the 80 kD polypeptide is cleaved again into a 20 kD and C-terminal 60 kD polypeptide. The full-length proprotein and the 25, 80 and 60 kD (s25, s80, s60) derivatives have been identified in the wild-type egg shell, implying that cleavage is an extracellular event. Immunogold analysis using these antisera indicate that the position of these proteins is dynamic during choriogenesis. Western blots of chorion and vitelline membrane indicates that by stage 14, s60 is found in both layers but the protein in the chorion has a higher mobility, implying that s60 undergoes some post-depositional modification. In addition, Dr. Waring has constructed a P-element vector containing the dec-1 gene and flanking DNA which is capable of full rescue of fs(1)410 dec-1 females. Using the Drosophila antibodies, she has shown that the post-depositional processing of the Dec-1 protein is conserved in D. virilis (50-80 Myr removed), although the sequence has diverged sufficiently to prevent cross-hybridization to a variety of D. melanogaster probes even at low stringency. Primer pairs from the more closely related D. pseudoobscura (20-50 Myr. removed) has allowed amplification of a single PCR product that hybridizes to RNA of the size and temporal specificity expected for dec-1 transrcipts in D. virilis. First, Dr. Waring proposes to characterize the post-depositional change responsible for the observed mobility shift in the s60 polypeptide. This polypeptide contains several potential phosphorylation sites. To determine if these sites are phosophylated Dr. Waring proposes to inject 32P into adult females and isolate proteins from stage 14 oocytes 8 hr. later. These proteins will be separated by electrophoresis and the s60 polypeptide identified by its position with respect to a Western blot of unlabeled protein run in parallel. Alternatively, she will determine if the electrophoretic heterogeneity is eliminated in response to treatment with alkaline phosphatase. In addition, Dr. Waring wants to determine which part of the s60 molecule is responsible for the mobility shift by generating polypeptide fragments using partial proteolysis or digestion with a protease. These fragments from chorion and vitelline membrane proteins will be separated on high resolution polyacrylamide gels and visualized by radiolabel or antibody staining. If the position of the modified residue can be determined using these methods, in-vitro mutagenesis and P-element transformation will be used to introduce s60 mutants unable to undergo modification into a dec-1-background. Analysis of the distribution of these polypeptides will elucidate whether the post-depositional modification is necessary for interlayer trafficking of this protein. Second, Dr. Waring would like to identify which motifs in the s60 peptide are important for interactions with the vitelline membrane and the chorion layers. In order to do this, she proposed to first isolate and characterize the D. virilis dec-1 gene. As part of this study she proposes to transform dec-1-D. melanogaster with the D. virilis gene and ask if the Dv s80 and s60 polypeptides are sequestered in the vitelline membrane and if the D. virilis gene can rescue the D. melanogaster chorion phenotype and restore female fertility. Dr. Waring will use these results to direct an in vitro mutagenesis study. If the Dv gene fully rescues the melanogaster phenotype, mutagenesis will concentrate on conserved regions. If the Dv product does not accumulate in the vitelline membrane, Dv-Dm chimeric proteins will be analyzed to determine which parts of the Dm protein are required for chorion localization. Third, Dr. Waring proposes to identify eggshell proteins which interact with dec-1 proteins. 35S-labeled s60 and s80 will be generated in either a prokaryotic or, if the loss of post-translational modifications proves problematic, in a Baculovirus system. These proteins will be used to probe egg-shell proteins from different stages separated by SDS-PAGE and blotted on PVDF filters. Previously known egg-shell proteins can be identified by their mobility on a 2-dimensional gel.