RESUMEN
Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex.
Asunto(s)
Factor 1 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica , Proteínas Fúngicas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor 1 Eucariótico de Iniciación/química , Factor 1 Eucariótico de Iniciación/aislamiento & purificación , Factor 5 Eucariótico de Iniciación , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Genotipo , Cinética , Modelos Moleculares , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/aislamiento & purificación , Factor 3 Procariótico de Iniciación , Biosíntesis de Proteínas , Subunidades de Proteína , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/aislamiento & purificación , Ribosomas/ultraestructura , TermodinámicaRESUMEN
eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3.