Protein 4.1R binding to eIF3-p44 suggests an interaction between the cytoskeletal network and the translation apparatus.
Erythroid protein 4.1 (4.1R) is an 80-kd cytoskeletal protein that stabilizes the membrane-skeletal network structure underlying the lipid bilayer. Using the carboxyl terminal domain (22/24 kd) of 4.1R as bait in a yeast 2-hybrid screen, we isolated cDNA clones encoding a polypeptide of eIF3-p44, which represents a subunit of a eukaryotic translation initiation factor 3 (eIF3) complex. The eIF3 complex consists of at least 10 subunits that play an essential role in the pathway of protein translation initiation. Northern blot analysis revealed that eIF3-p44 (approximately 1.35 kb) is constitutively expressed in many tissues. The essential sequence for this interaction was mapped to the carboxyl-terminus of 4.1R (residues 525-622) and a region (residues 54-321) of eIF3-p44. The direct association between 4.1R and eIF3-p44 was further confirmed by in vitro binding assays and coimmunoprecipitation studies. To characterize the functions of eIF3-p44, we depleted eIF3-p44 from rabbit reticulocyte lysates either by anti-eIF3-p44 antibody or by GST/4.1R-80 fusion protein. Our results show that the eIF3-p44 depleted cell-free translation system was unable to synthesize proteins efficiently. The direct association between 4.1R and elF3-p44 suggests that 4.1R may act as an anchor protein that links the cytoskeleton network to the translation apparatus. (Blood. 2000;96:747-753)
Hou
C L
CL
Institute of Life Science, National Defense Medical Center, and the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Tang
C j
Cj
Roffler
S R
SR
Tang
T K
TK
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D002474:Cell-Free System; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009479:Neuropeptides; D009693:Nucleic Acid Hybridization; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000906:Antibodies; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D011993:Recombinant Fusion Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D005982:Glutathione Transferase