Intracellular targeting and mRNA interactions of the eukaryotic translation initiation factor eIF4E in the yeast Saccharomyces cerevisiae.
The 5' cap structure of eukaryotic mRNAs is believed to play a role in a number of cellular processes, including pre-mRNA splicing, nuclear export and translation. An essential cap-binding protein that is likely to mediate the participation of the cap in at least one of these processes is the eukaryotic translation initiation factor eIF4E. This protein is thought to facilitate the initial ribosomal interaction with the 5' end of the mRNA, involving the binding of eIF4E to the cap in the cytoplasm. Yet the subcellular distribution and mechanism of targeting of eIF4E has been an unresolved issue. We have therefore examined whether eIF4E in the yeast Saccharomyces cerevisiae is directed to the nucleus by virtue of a nuclear localization sequence (NLS) in its amino acid sequence. eIF4E was fused with the "marker proteins' yeast invertase and jellyfish green fluorescent protein. The distribution of these fusions could be followed using immunofluorescence and confocal microscopy of protoplasts and whole cells. These and other fusions were used to show that while yeast eIF4E does not possess an efficiently functioning NLS, it can be transported into the nucleus if provided with a known active NLS. However, an NLS-eIF4E fusion of this type cannot be stably supported by the cell, most likely because of its inhibitory effects when present in large quantities in the nucleus, whereas an NLS fusion with a mutant form of eIF4E that has reduced cap-affinity is tolerated.
Ptushkina
M
M
Department of Gene Expression, National Biotechnology Research Centre, Braunschweig, Germany.
Vasilescu
S
S
Fierro-Monti
I
I
Rohde
M
M
McCarthy
J E
JE
D001483:Base Sequence; D015415:Biomarkers; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012315:RNA Caps / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D015415:Biomarkers; D039561:Eukaryotic Initiation Factor-4E; D008164:Luminescent Proteins; D010448:Peptide Initiation Factors; D012315:RNA Caps; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase