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PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes.

Zhou C, Arslan F, Wee S, Krishnan S, Ivanov AR, Oliva A, Leatherwood J, Wolf DA - BMC Biol. (2005)

Bottom Line: Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation.Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes.We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA. czhou@rics.bwh.harvard.edu

ABSTRACT

Background: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.

Results: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis.

Conclusion: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

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Distinct eIF3 complexes. (A) eIF3 complexes associated with eIF3e and eIF3m were affinity-purified by absorption of the respective pro-A-tagged proteins to IgG beads. Bound proteins were eluted by cleavage with SDS (eIF3e) or TEV protease (eIF3m). (B) The eluates described in (A) were analyzed by immunoblotting with eIF3d antibodies. The asterisk refers to cross-reactivity of the secondary antibody with Ig light chains. (C) Lysates from cells expressing Myc-tagged Csn5p, eIF3e, and eIF3m from their endogenous genomic loci were immunoprecipitated with anti-eIF3d antisera. Coprecipitated proteins were identified by immunoblotting as indicated. Total cell lysates are shown to document expression levels of the endogenously tagged proteins. (D) Protein lysate from cells expressing protein A-tagged eIF3b from the endogenous promoter was absorbed to IgG beads, and specifically retained proteins were identified by LC-MS/MS.
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Figure 3: Distinct eIF3 complexes. (A) eIF3 complexes associated with eIF3e and eIF3m were affinity-purified by absorption of the respective pro-A-tagged proteins to IgG beads. Bound proteins were eluted by cleavage with SDS (eIF3e) or TEV protease (eIF3m). (B) The eluates described in (A) were analyzed by immunoblotting with eIF3d antibodies. The asterisk refers to cross-reactivity of the secondary antibody with Ig light chains. (C) Lysates from cells expressing Myc-tagged Csn5p, eIF3e, and eIF3m from their endogenous genomic loci were immunoprecipitated with anti-eIF3d antisera. Coprecipitated proteins were identified by immunoblotting as indicated. Total cell lysates are shown to document expression levels of the endogenously tagged proteins. (D) Protein lysate from cells expressing protein A-tagged eIF3b from the endogenous promoter was absorbed to IgG beads, and specifically retained proteins were identified by LC-MS/MS.

Mentions: Like the eIF3m complex, the eIF3e complex contained roughly stoichiometric amounts of the eIF3 core subunits a, b, c, g, and i (Fig. 3A, Table 1). In addition, eIF3f was present while, unexpectedly, eIF3m and eIF3h were missing. We did not detect the proteasome lid subunit Rpn5p, which previously had been shown to associate with eIF3e and eIF3d [45]. This observation indicated that the described eIF3d/eIF3e/Rpn5p interaction is either unstable under our purification conditions or contains only a minor fraction of the total eIF3e engaged in protein interactions.


PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes.

Zhou C, Arslan F, Wee S, Krishnan S, Ivanov AR, Oliva A, Leatherwood J, Wolf DA - BMC Biol. (2005)

Distinct eIF3 complexes. (A) eIF3 complexes associated with eIF3e and eIF3m were affinity-purified by absorption of the respective pro-A-tagged proteins to IgG beads. Bound proteins were eluted by cleavage with SDS (eIF3e) or TEV protease (eIF3m). (B) The eluates described in (A) were analyzed by immunoblotting with eIF3d antibodies. The asterisk refers to cross-reactivity of the secondary antibody with Ig light chains. (C) Lysates from cells expressing Myc-tagged Csn5p, eIF3e, and eIF3m from their endogenous genomic loci were immunoprecipitated with anti-eIF3d antisera. Coprecipitated proteins were identified by immunoblotting as indicated. Total cell lysates are shown to document expression levels of the endogenously tagged proteins. (D) Protein lysate from cells expressing protein A-tagged eIF3b from the endogenous promoter was absorbed to IgG beads, and specifically retained proteins were identified by LC-MS/MS.
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Figure 3: Distinct eIF3 complexes. (A) eIF3 complexes associated with eIF3e and eIF3m were affinity-purified by absorption of the respective pro-A-tagged proteins to IgG beads. Bound proteins were eluted by cleavage with SDS (eIF3e) or TEV protease (eIF3m). (B) The eluates described in (A) were analyzed by immunoblotting with eIF3d antibodies. The asterisk refers to cross-reactivity of the secondary antibody with Ig light chains. (C) Lysates from cells expressing Myc-tagged Csn5p, eIF3e, and eIF3m from their endogenous genomic loci were immunoprecipitated with anti-eIF3d antisera. Coprecipitated proteins were identified by immunoblotting as indicated. Total cell lysates are shown to document expression levels of the endogenously tagged proteins. (D) Protein lysate from cells expressing protein A-tagged eIF3b from the endogenous promoter was absorbed to IgG beads, and specifically retained proteins were identified by LC-MS/MS.
Mentions: Like the eIF3m complex, the eIF3e complex contained roughly stoichiometric amounts of the eIF3 core subunits a, b, c, g, and i (Fig. 3A, Table 1). In addition, eIF3f was present while, unexpectedly, eIF3m and eIF3h were missing. We did not detect the proteasome lid subunit Rpn5p, which previously had been shown to associate with eIF3e and eIF3d [45]. This observation indicated that the described eIF3d/eIF3e/Rpn5p interaction is either unstable under our purification conditions or contains only a minor fraction of the total eIF3e engaged in protein interactions.

Bottom Line: Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation.Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes.We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, USA. czhou@rics.bwh.harvard.edu

ABSTRACT

Background: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.

Results: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis.

Conclusion: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

Show MeSH
Related in: MedlinePlus