The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.
The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Osvaldo P
OP
de Melo Neto
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Tamara D C
TD
da Costa Lima
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Camila C
CC
Xavier
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.|b CHU de Quebec Research Center and Department of Microbiology ; Infectious Disease and Immunology; Laval University ; Quebec, QC , Canada.
Larissa M
LM
Nascimento
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Tatiany P
TP
Romão
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Ludmila A
LA
Assis
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Mariana M C
MM
Pereira
a Departamento de Microbiologia ; Centro de Pesquisas Aggeu Magalhães-FIOCRUZ ; Recife , PE , Brazil.
Christian R S
CR
Reis
b CHU de Quebec Research Center and Department of Microbiology ; Infectious Disease and Immunology; Laval University ; Quebec, QC , Canada.
Barbara
B
Papadopoulou
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D007891:Leishmania / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D039102:Poly(A)-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D016415:Sequence Alignment
D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039102:Poly(A)-Binding Proteins
Leishmania; cap binding protein; eIF4E; eIF4G; phosphorylation; protein synthesis; translation initiation PABP