IDENTIFICATION OF PROTEIN-PROTEIN INTERACTIONS OF THE MAJOR SPERM PROTEIN (MSP) OF CAENORHABDITIS-ELEGANS

In nematodes, sperm are amoeboid cells that crawl via an extended pseu dopod. Unlike those in other crawling cells, this pseudopod contains l ittle or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitvo studies of Ascaris suum MSP have demonstrated th at motility occurs via the regulated assembly and disassembly of MSP f ilaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investig ate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ repo rter construct. A library of AD-MSP mutants was generated via mutageni c PCR and screened for clones that fail to interact with LEXA-MSP. Sin gle missense mutations were identified and mapped to the crystal struc ture of A. suum MSP. Two classes of mutations predicted from the struc ture were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface . Multiple additional mutations were obtained in the two carboxy-termi nal beta strands, a region not predicted to be involved in protein fol ding or dimer formation. Size fractionation of bacterially expressed M SPs indicates that mutations in this region do not abolish dimer forma tion. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal be ta strands are directly involved in interactions required for MSP fila ment assembly. (C) 1998 Academic Press Limited.