Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis.
We have examined the mechanism of membrane protein insertion in the ameboid spermatozoa of Caenorhabditis elegans using two monoclonal antibodies which recognize the same set of eight sperm-specific polypeptides. Previous electron microscopic studies demonstrated that these antibodies label surface and cytoplasmic populations of antigen. Cells whose surface antigen had been removed by proteolysis were able to localize new membrane protein insertion at the tips of pseudopodial projections. C. elegans sperm do not contain the protein synthesizing machinery needed for delivery of new membrane to the cell surface. It has, therefore, been of interest to determine how localized membrane assembly occurs. Here we have determined the subcellular location of each of these eight polypeptides. A closely positioned doublet of bands around 97 kD (comprising 40% of the total antigen in sperm) represents surface (larger member of doublet) and cytoplasmic (lower member) forms of protein. Proteolysis of live cells eliminated this surface form from immunoblots but did not affect the cytoplasmic protein. When cells were allowed to reinsert new protein following removal of the enzyme, this surface form was regenerated. Since sperm are unable to synthesize new protein, this higher molecular weight species may arise from a posttranslational modification of proteins in the cytoplasmic pool. We present evidence suggesting that the surface protein is generated from this cytoplasmic pool by addition of fatty acid. Fatty acid acylation would account for both the observed decrease in electrophoretic mobility of the surface form and provide increased hydrophobicity to the protein which may allow for its insertion into the lipid bilayer.
Pavalko
F M
FM
Department of Biological Science, Florida State University, Tallahassee 32306.
Roberts
T M
TM
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D002107:Caenorhabditis / Q000166:cytology*; D002465:Cell Movement / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism* / Q000648:ultrastructure; D010738:Type C Phospholipases
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008565:Membrane Proteins; D010738:Type C Phospholipases