Identification of neighboring protein pairs in the 60 S ribosomal subunits from Saccharomyces cerevisiae by chemical cross-linking.
Protein-protein cross-linking was used to determine the spatial arrangement of proteins within the 60 S ribosomal subunits of Saccharomyces cerevisiae. Protein cross-links were generated by treatment of intact ribosomal subunits with dimethyl 3,3'-dithiobispropionimidate. Proteins were extracted from the treated subunits and fractionated by Cm-cellulose chromatography. Cross-linked proteins in these fractions were analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing sodium dodecyl sulfate. Component members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Seventeen pairs involving 16 of the 45 60 S subunit proteins were identified. Several proteins were detected in numerous cross-linked dimers and were used as foci for constructing a model depicting the arrangement of proteins within the 60 S ribosomal subunit. The model also incorporated previously published data on structure and function of proteins from the yeast 60 S subunit.
Xiang
R H
RH
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Lee
J C
JC
D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D007096:Imidoesters; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008961:Models, Structural; D008970:Molecular Weight; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000032:analysis* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D013388:Succinimides
D003432:Cross-Linking Reagents; D007096:Imidoesters; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D012269:Ribosomal Proteins; D013388:Succinimides; C007471:dimethyl dithiobispropionimidate; C022581:Bolton-Hunter reagent