Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E.
Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.
Fierro-Monti
Ivo
I
Department of Biochemistry and Molecular Biology, University of Southern Denmark. ivofm@bmb.sdu.dk
Mohammed
Shabaz
S
Matthiesen
Rune
R
Santoro
Raffaela
R
Burns
Jorge S
JS
Williams
David J
DJ
Proud
Christopher G
CG
Kassem
Moustapha
M
Jensen
Ole N
ON
Roepstorff
Peter
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D012315:RNA Caps / Q000502:physiology; D020871:RNA Stability; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D055532:SMN Complex Proteins
D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; C449808:GEMIN5 protein, human; D012315:RNA Caps; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins