Abstract
BackgroundThe C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure.ResultsWe made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants.ConclusionsThe results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.
Highlights
The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization
We developed a structural model of SPE-42 using the structures of known RING finger containing proteins
The model is based on sequence conservation with the four most closely related RING finger sequences in the RSCB protein data bank (PDB) http://www.rcsb.org/pdb/home/home.do; c-Cbl protooncogene RING domain [pbd:1FBV] [42], tumor necrosis factor receptor-associated factor-6 (TRAF-6) RING domain [pdb:3HCT] [40], CCR4-NOT transcription complex, subunit 4, (CNOT4) [pdb:1UR6] [43], and cellulose synthase, catalytic subunit (IRX3), [pdb:1WEO] (He, F., et al personal communication)
Summary
The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. The five cloned genes in this class have been shown, or are predicted, to be sperm plasma membrane proteins [13,16,17,18,19]. The phenotype of these mutants suggests that spe-42 and the other spe-9 class genes function at the moment that sperm and egg plasma membranes meet
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