RESEARCH 2002
RESEARCH 2001
RESEARCH 2000

Megan Mayerle
Marquette University
Milwaukee, WI
Mentor: Dr. Gail Waring

The Drosophila eggshell is a complex multilayered structure that forms in late oogenesis between the oocyte and the overlying follicle cells. The protein on which my research focuses is the sV23 protein located in the vitelline membrane (Vm) of the eggshell. The sV23 protein is both synthesized and secreted in stages 9 to 10 of egg chamber development. At stage 10b C-terminal processing of sV23 occurs in the eggshell followed by N-terminal processing during stages 12-13. sV23 is a Vm protein and thus it contains the characteristic Vm domain that includes three cysteine residues involved in disulfide bond formation. The disulfide network that forms within the vitelline membrane layer of the eggshell appears to enlarge during the later stages of oogenesis as eggshell development proceeds. 

The purpose of my project is to gain some insight into the protein-protein interactions that occur within the vitelline membrane, particularly which Vm proteins form disulfide bonds with each other. Vm proteins interact through both covalent and non-covalent interactions. In order to determine what is necessary to release sV23 from the eggshell, egg chambers at various stages were exposed to various solubilizing conditions and pelleted at 15,000 x g. The supernatant and pellet fractions were then analyzed by Western Blot analysis. I also utilized sV23 C2,3‡S mutants, where a single cysteine residue remains in sV23’s Vm domain, to determine if sV23 bonds to more than one Vm protein.

In wild type flies I have shown that sV23 can be released from the eggshell during stage 10 of egg chamber development using different solubilizing agents such as Urea and SDS, however these same agents fail to release sV23 in stage 12-14 egg chambers. A weaker non-ionic detergent, Triton X-100, was unable to release sV23 even in stage 10 egg chambers. This suggests only a minimal disruption of non-covalent interactions in the eggshell with Triton X-100. In contrast, including _ME, which breaks disulfide bonds in the initial solubilization of the egg chambers, released the sV23 protein into the supernatant at all stages of egg chamber development tested regardless of the solubilizing agent used. These findings indicate that solubilizing agents that disrupt only non-covalent interactions are insufficient to release sV23 once it has been incorporated into a larger disulfide bonded network in later stage egg chambers.

In sV23 C2,3‡S mutant egg chambers solubilized with Triton X-100, SDS, or Urea, sV23 is found in the supernatant fraction at all stages even in the absence of _ME. This indicates that, unlike the wild type fly, the mutant fly is not able to form complexes involving sV23 that are large enough to be pelleted at any stage. To determine what sV23 is forming disulfide bonds with, C2,3‡S mutant egg chambers were solubilized in SDS and loaded onto a gel in the absence of _ME and analyzed by Western Blot analysis. Multiple bands were seen indicating that sV23 forms disulfide-bonded complexes with other Vm protein(s).