IDENTIFICATION OF CDNA CLONES ENCODING VALOSIN-CONTAINING PROTEIN ANDOTHER PLANT PLASMA MEMBRANE-ASSOCIATED PROTEINS BY A GENERAL IMMUNOSCREENING STRATEGY

An approach was developed for the isolation characterization of soybea n plasma membrane-associated proteins by immunoscreening of a cDNA exp ression library. An antiserum was raised against purified plasma membr ane vesicles, In a differential screening of approximate to 500,000 pl aque-forming units with the anti-(plasma membrane) serum and DNA probe s derived from highly abundant clones isolated in a preliminary screen ing, 261 clones were selected from approximate to 1,200 antiserum-posi tive plaques. These clones were classified into 40 groups by hybridiza tion analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identif ied, among which valosin-containing protein (VCP), clathrin heavy chai n, phospholipase C, and sylmethionine:Delta(24)-sterol-C-methyltransfe rase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, repre sent additional or yet uncharacterized genes. A full-length cDNA encod ing the soybean VCP was sequenced. The high level of amino acid identi ty with vertebrate VCP and yeast CDC48 protein indicates that the soyb ean protein is a plant homolog of vertebrate VCP and yeast CDC48 prote in.