Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.
An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.
Shi
J
J
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Dixon
R A
RA
Gonzales
R A
RA
Kjellbom
P
P
Bhattacharyya
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015342:DNA Probes; D018076:DNA, Complementary; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism*; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D015342:DNA Probes; D018076:DNA, Complementary; D018744:DNA, Plant; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D010940:Plant Proteins; C046951:valosin; D000074405:Valosin Containing Protein; C093549:Vcp protein, Glycine max