Translation initiation in Leishmania major: characterisation of multiple eIF4F subunit homologues.
In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F - eIF4E, eIF4A and eIF4G - to the monomethylated cap present on the 5' end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5' end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae. However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5' ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es (LmEIF4E1-4), two eIF4As (LmEIF4A1-2) and five eIF4Gs (LmEIF4G1-5). We report the initial characterisation of LmEIF4E1-3, LmEIF4A1-2 and LmEIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. LmEIF4A1 and LmEIF4E3 are very abundant, LmEIF4G3 is moderately abundant and LmEIF4E1/LmEIF4E2/LmEIF4A2 are rare or not detected. In cap-binding assays, only LmEIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that LmEIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A (LmEIF4A1/LmEIF4A2) and eIF4G (LmEIF4G1-3) homologues. Only LmEIF4G3, via the HEAT domain, bound specifically both to LmEIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.
Dhalia
Rafael
R
Departamento de Biologia Celular, Universidade de Brasilia, Brasilia 70910-900, D.F., Brazil.
Reis
Christian R S
CR
Freire
Eden R
ER
Rocha
Pollyanna O
PO
Katz
Rodolfo
R
Muniz
João R C
JR
Standart
Nancy
N
de Melo Neto
Osvaldo P
OP
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D039562:Eukaryotic Initiation Factor-4F / Q000096:biosynthesis* / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011506:Proteins; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism; D016415:Sequence Alignment; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D018797:Cell Cycle Proteins; D039562:Eukaryotic Initiation Factor-4F; D047908:Intracellular Signaling Peptides and Proteins; D021122:Protein Subunits; D011506:Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D000099259:N-myc Downstream-Regulated Gene 1 Protein