Conserved Cysteines in the Vitelline Membrane Domain of the Drosophila sV23 Eggshell Protein are Functionally Distinct

The vitelline membrane (VM) of the Drosophila eggshell is composed of approximately six major proteins (VMPs) that possess a conserved VM domain which includes three precisely spaced, evolutionarily conserved, cysteines (CX\u27CX C). sV23, a major VMP essential for female fertility, was studied to investigate how proteins assemble into a functional extracellular structure in vivo. By making a series of cysteine-to-serine substitution mutations at different positions and in different combinations, we have shown that the three cysteines (CI23, CI31 and CI40) are not functionally equivalent. While mutations on the first (C123S) or third cysteine (C140S) were tolerated, a mutation at the second position (CI3IS) impaired its function suggesting that C131 is a critical residue. In double cysteine mutants, when the second cysteine (CI31) is retained in sV23, females have aberrant fertility, which indicates that CI31 is not sufficient for the function of sV23. In wild type, sV23 is incorporated into a large disulfide network at stage 13 of oogenesis. Like wild type, sV23 in all three single substitution mutants was incorporated into a large disulfide linked network. In contrast, sV23 in double cysteine mutants failed to incorporate into a large disulfide network when C131 was not retained. The formation of non-reducible cross-link His-tagged wild type and mutant sV23 proteins were used to isolate disulfide linked sV23 containing VMP complexes by Nickel affinity chromatography. VM protein sV58 and sV17 were found in the same disulfide network as sV23 in the early stage of eggshell formation. The complexity of the sV23 disulfide network in a double cysteine mutant C123S/C131S seemed comparable to wild type during the early stages. However unlike wild type, a significant proportion of other VMPs were not included in the mutant sV23 complexes and sV23 was recovered predominately as a putative homodimer. In contrast to initial assembly, the association of sV23 with sV17 was lost at later stages suggesting that reshuffling of disulfide bonds may occur during the assembly of the VM network