Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system.
In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B gamma. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein-protein interactions. The following complexes were observed: eEF1A1:eEF1B alpha, eEF1A1:eEF1B beta, eEF1B beta:eEF1B beta, eEF1B alpha:eEF1B gamma, eEF1B beta:eEF1B gamma and eEF1B alpha:eEF1B gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1B alpha or eEF1B beta held together by eEF1B gamma. These units can dimerize via eEF1B beta. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.
Mansilla
Francisco
F
Institute of Molecular and Structural Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Arhus C, Denmark.
Friis
Irene
I
Jadidi
Mandana
M
Nielsen
Karen M
KM
Clark
Brian F C
BF
Knudsen
Charlotte R
CR
D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms; D021122:Protein Subunits; D020798:Two-Hybrid System Techniques
D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D020033:Protein Isoforms; D021122:Protein Subunits; C057342:peptide elongation factor 1H