The polypyrimidine tract binding protein is a monomer.
The polypyrimidine tract binding (PTB) protein is a potent regulator of alternative mRNA splicing. It also participates in other essential cellular functions, including translation initiation and polyadenylation. Several published reports have suggested that the protein forms a dimer in solution, a feature that has been widely incorporated into mechanistic models of protein function. However, recent studies have provided indications that full-length PTB is a monomer. Here we present new biophysical and biochemical evidence supporting the monomeric status of the protein. By use of blue-native polyacrylamide gel electrophoresis and size-exclusion chromatography, PTB was observed as a single molecular species under native reducing environments, though in oxidizing conditions, a larger protein species was also detected. Further analyses of wild-type and mutant PTB molecules with SDS-PAGE and time-of-flight electrospray ionization mass spectroscopy confirmed these observations. They also identified the single reduced species as monomeric PTB and the higher-molecular-weight nonreduced species as disulphide-linked PTB dimer mediated by Cys23. Our results indicate that the use of oxidizing environments in previous studies is likely to have contributed to the mis-assignment of PTB as a dimer. Although purified PTB may form disulphide-linked dimers under these conditions, in the reducing intracellular environment the protein will be monomeric. These findings have implications for the construction of models of PTB function in regulating mRNA metabolism.
Monie
Tom P
TP
Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom.
Hernandez
Helena
H
Robinson
Carol V
CV
Simpson
Peter
P
Matthews
Stephen
S
Curry
Stephen
S
D017398:Alternative Splicing; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D004220:Disulfides; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; D003545:Cysteine