Skip to main content
Figure 2 | BMC Biology

Figure 2

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

Figure 2

Roles of eif3e and eif3m in protein synthesis. (A) Subcellular localization. Live cells expressing GFP-tagged alleles of eif3b, eif3e, and eif3m at the endogenous genomic loci were examined by fluorescence microscopy. N-terminally GFP-tagged eif3f was expressed at low levels from the pREP81 plasmid. (B) Shut-off strains. Diploid heterozygous eif3f and eif3m deletion strains were transformed with pREP81 plasmids driving the thiamin-repressible expression of Myc-tagged eIF3f and eIF3m, respectively. Diploids were sporulated and homozygous disruptants carrying the eif3f and eif3m plasmids were selected. Cells were grown in liquid medium to an OD595 of 0.3 in the absence of thiamin, followed by promoter shut-off by the addition of thiamin. Samples were taken at the indicated time points after promoter shut-off and analyzed for the expression of plasmid borne eIF3f and eIF3m by immunoblotting with anti-Myc antibodies. (C) Effect on total protein synthesis. The eif3f and eif3m shut-off strains were maintained in the absence or presence of thiamin as indicated. Strains transformed with empty pREP81 plasmid were included as control. Cells were metabolically labeled with 35S-methionine, and aliquots of total cellular proteins were separated by SDS-PAGE and analyzed by autoradiography. The Coomassie Blue stained gel is shown to document equal protein loading. Data were quantified by PhosphorImager analysis and results are displayed in a bar graph. (D) Effect on polysomes. Polysome profiles were determined for the indicated strains as described in the Methods section. An eif3e deletion strain is shown for reference. (E) Effect on CSN-mediated regulation of cullin-RING ubiquitin ligases. Cul1p complexes were immunopurified from the indicated strains and employed in substrate-independent in vitro ubiquitylation reactions with purified E1, the E2 Cdc34p, ubiquitin, and ATP [26]. Polyubiquitin chains formed in the reaction are indicated (top panel). In contrast to csn5 mutants, derepression of Cul1p activity is not observed in cells lacking eif3f, eif3m, or eif3e. The neddylation state of Cul1p was determined by immunoblotting (middle panel). Hyperneddylated Cul1p only accumulates in csn5 mutants. Shut-off of eif3f and eif3m expression was verified by immunoblotting with anti-Myc antibodies (lower panel).

Back to article page