title: Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, Swenson College of Science and Engineering, University of Minnesota Duluth, Duluth, MN 55812, USA.
authors.forename: Luke D
authors.initials: LD
authors.lastname: Wilson
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Sackett
authors.forename: Bryce D
authors.initials: BD
authors.lastname: Mieczkowski
authors.forename: Abigail L
authors.initials: AL
authors.lastname: Richie
authors.forename: Kara
authors.initials: K
authors.lastname: Thoemke
authors.forename: Jon N
authors.initials: JN
authors.lastname: Rumbley
authors.forename: Tim L
authors.initials: TL
authors.lastname: Kroft
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005306:Fertilization; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D054829:RING Finger Domains / Q000235:genetics; D020539:Sequence Analysis, Protein; D013084:Sperm-Ovum Interactions; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry
chemical_list: D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; C505795:spe-42 protein, c elegans; D015032:Zinc; D003545:Cysteine