Mass spectrometric analysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms.
In eukaryotes, translation initiation factor 4G (eIF4G) acts as the central binding protein for an unusually large number of proteins involved in mRNA metabolism. Several gene products homologous to eIF4G have been described, the most studied being eIF4G-1. By its association with other initiation factors, eIF4G-1 effects mRNA cap and poly(A) recognition, unwinding of secondary structure, and binding to the 43S initiation complex. Multiple electrophoretic isoforms of eIF4G-1 are observed, and multiple cDNAs have been reported, yet the relationship between the two is not known. We report here a new cDNA for eIF4G-1, present as a previously unidentified human expressed sequence tag, that extends the long open reading frame, provides a new in-frame initiation codon, and predicts a longer form of eIF4G-1 than reported previously. eIF4G isoforms from human K562 cells were cleaved with recombinant Coxsackievirus 2A protease and the N- terminal domains purified by m(7)GTP-Sepharose chromatography and polyacrylamide gel electrophoresis. Proteins were digested with proteolytic enzymes and peptides masses determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In selected cases, peptides were sequenced by electrospray-mass spectrometry fragmentation. This identified the N termini of the three most abundant eIF4G-1 isoforms, two of which had not previously been proposed. These proteins appear to have been initiated from three different AUG codons.
Bradley
Christopher A
CA
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.
Padovan
Júlio César
JC
Thompson
Timothy L
TL
Benoit
Clint A
CA
Chait
Brian T
BT
Rhoads
Robert E
RE
D000595:Amino Acid Sequence; D039603:Eukaryotic Initiation Factor-4G; D020224:Expressed Sequence Tags; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C113920:EIF4G1 protein, human; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D020033:Protein Isoforms; D012333:RNA, Messenger