Drosophila vitelline membrane assembly: a critical role for an evolutionarily conserved cysteine in the "VM domain" of sV23.
The vitelline membrane (VM), the oocyte proximal layer of the Drosophila eggshell, contains four major proteins (VMPs) that possess a highly conserved "VM domain" which includes three precisely spaced, evolutionarily conserved, cysteines (CX⁷CX⁸C). Focusing on sV23, this study showed that the three cysteines are not functionally equivalent. While substitution mutations at the first (C123S) or third (C140S) cysteines were tolerated, females with a substitution at the second position (C131S) were sterile. Fractionation studies showed that sV23 incorporates into a large disulfide linked network well after its secretion ceases, suggesting that post-depositional mechanisms are in place to restrict disulfide bond formation until late oogenesis, when the oocyte no longer experiences large volume increases. Affinity chromatography utilizing histidine tagged sV23 alleles revealed small sV23 disulfide linked complexes during the early stages of eggshell formation that included other VMPs, namely sV17 and Vml. The early presence but late loss of these associations in an sV23 double cysteine mutant suggests that reorganization of disulfide bonds may underlie the regulated growth of disulfide linked networks in the vitelline membrane. Found within the context of a putative thioredoxin active site (CXXS) C131, the critical cysteine in sV23, may play an important enzymatic role in isomerizing intermolecular disulfide bonds during eggshell assembly.
Wu
T
T
Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.
Manogaran
A L
AL
Beauchamp
J M
JM
Waring
G L
GL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D019143:Evolution, Molecular; D005260:Female; D017344:Genes, Insect; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009866:Oogenesis / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014817:Vitelline Membrane / Q000254:growth & development* / Q000378:metabolism*
D017931:DNA Primers; D029721:Drosophila Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C487462:sV23 protein, Drosophila; D003545:Cysteine