title: Fusion proteins ofArabidopsiscap-binding proteins: Cautionary "tails" of woe.
abstract: The use of fluorescent proteins fused to other proteins has been very useful in revealing the location and function of many proteins. However, it is very important to show that the fusion of these reporter proteins does not impact the function of the protein of interest. Plants have 2 forms of the cap-binding protein that function in initiation of translation, eIF4E and a plant specific form, eIFiso4E. In an attempt to determine the cellular localization of eIFiso4E, fusions to GFP were made, but were found to not be competent to rescue the lethal phenotype of plants lacking eIF4E and eIFiso4E. This suggested that the GFP fusions at either the N- or C-terminus of eIFiso4E were not functional. Biochemical analysis of the fusions revealed that eIFiso4E•GFP fusions were not able to bind to m7GTP Sepharose indicating that they were not functional as cap-binding proteins. Analysis of eIF4E•GFP fusions, both in yeast andin vitro, showed that the N-terminal fusion may be functional, whereas the C-terminal fusion bound m7GTP Sepharose very poorly and functioned poorly in yeast. These results highlight the importance of verification both biochemically andin vivothat reporter fusions of proteins maintain activity and are stable in order to prevent observations that may result in artifacts.
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Levins
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Ching-Ying
authors.initials: CY
authors.lastname: Tseng
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Patrick
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Mayberry
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Nicola
authors.initials: N
authors.lastname: Cole
authors.affiliation: Department of Molecular Biosciences and Institute for Cell and Molecular Biology, University of Texas at Austin , Austin, TX, USA.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Browning
keywords: GFP; cap-binding protein; eukaryotic initiation factor; fusion protein; translation