Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

• Published in: BMC developmental biology 2011-02, Vol.11 (1), p.10-10, Article 10
• Main Authors: Wilson, Luke D, Sackett, Jacqueline M, Mieczkowski, Bryce D, Richie, Abigail L, Thoemke, Kara, Rumbley, Jon N, Kroft, Tim L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Caenorhabditis elegans; Caenorhabditis elegans - genetics; Caenorhabditis elegans - physiology; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - metabolism; Cysteine - chemistry; Cysteine - metabolism; Fertilization; Fertilization (Biology); Membrane Proteins - chemistry; Membrane Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Mutation; Physiological aspects; Polymerase Chain Reaction; Protein Interaction Domains and Motifs; Protein Structure, Tertiary; RING Finger Domains - genetics; Sequence Analysis, Protein; Sperm-Ovum Interactions; Spermatozoa; Structure-Activity Relationship; Ubiquitin-proteasome system; Zinc - chemistry; Zinc finger proteins
• ISSN: 1471-213X; 1471-213X
• DOI: 10.1186/1471-213X-11-10

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. 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. We 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. The 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.