Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

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In contrast to the knowledge regarding the function of chimeric Ewing sarcoma (EWS) fusion proteins that arise from chromosomal translocation, the cellular function of the RNA binding EWS protein is poorly characterized. EWS protein had been found mainly in the nucleus. In this report we show that EWS protein is not only found in the nucleus and cytosol but also on cell surfaces. After cell-surface biotinylation, isoelectric focusing of membrane fraction, avidin-agarose extraction of biotinylated proteins, and SDS-polyacrylamide gel electrophoresis, EWS protein was identified by matrixassisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of in-gel-digested peptides. These analyses revealed that the protein, having repeated RGG motifs, is extensively asymmetrically dimethylated on arginine residues, the sites of which have been mapped by mass spectrometric methods. Out of a total of 30 Arg-Gly sequences, 29 arginines were found to be at least partially methylated. The Arg-Gly-Gly sequence was present in 21 of the 29 methylation sites, and in contrast to other methylated proteins, only 11 (38%) methylated arginine residues were found in the Gly-Arg-Gly sequence. The presence of Gly on the C-terminal side of the arginine residue seems to be a prerequisite for recognition by a protein-arginine N-methyltransferase (PRMT) catalyzing this asymmetric dimethylation reaction. One monomethylarginine and no symmetrically methylated arginine residue was found. The present findings imply that RNA-binding EWS protein shuttles from the nucleus to the cell surface in a methylated form, the role of which is discussed.While investigating a 90-kDa anti-cyclophilin (anti-CyP) 1 immunoreactive band we noticed that anti-CyP antibodies recognized the RNA-binding Ewing sarcoma (EWS) protein and not a cyclophilin. The EWS gene is involved in tumor-related chromosomal translocations that associate part of EWS gene with various genes encoding transcription factors (1). The Nterminal transcriptional activation domain of EWS is fused to C-terminal DNA binding domains of corresponding partners. The translocation produces chimeric EWS proteins with transforming potential (2-7). The EWS gene of Ewing sarcoma and primitive neuroectodermal tumor is translocated to one of different members of the ETS (erythroblastosis virus-transforming sequence) family that contains the highly conserved DNA binding ETS domain. Often the ETS domain is derived from FLI-1 (Friend leukemia integration-1) and in rare cases from ERG (ETS-related gene), ETV-1 (ETS translocation variant-1), E1AF (E1A factor), or FEV (fifth Ewing variant). In malignant melanoma of soft parts, EWS is fused to ATF-1, in intraabdominal desmoplasmic small round-cell tumor to WT-1, in myxoid liposarcoma to CHOP, and in myxoid chrondrosarcoma to CHN (8).The cellular role of wild-type EWS protein remains less clear. The EWS protein is a nuclear protein with unknown function containing a C-terminal RNA binding motif and a N-terminal activation domain (9 -11). The IQ d...