Mass spectrometric analysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms

被引:39
作者
Bradley, CA
Padovan, JC
Thompson, TL
Benoit, CA
Chait, BT
Rhoads, RE
机构
[1] Louisiana State Univ, Hlth Sci Ctr, Dept Biochem & Mol Biol, Shreveport, LA 71130 USA
[2] Louisiana State Univ, Hlth Sci Ctr, Res Core Facil, Shreveport, LA 71130 USA
[3] Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, New York, NY 10021 USA
关键词
D O I
10.1074/jbc.M111134200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:12559 / 12571
页数:13
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