Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes

Abstract

Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

Figure 1.

Cluster analysis of ribosome fractions. (A) Purification schemes used to purify ribosomes and ribosome-associated proteins. (B) Clustering of proteins identified in ribosome purifications. Heat map clustering proteins by their PAF within functional categories (40S, 60S, translation related). The most abundant proteins are bright red and cluster at the top, yellow is intermediate, and black indicates absence of a particular protein. Each column represents an independent purification, and each row represents an individual protein.

Figure 2.

Identification of novel TMA proteins. Cluster analysis showing the relative abundances (PAFs) of uncharacterized ORFs identified in sucrose gradient fractions (A), total ribosome analysis (B), or ribosome salt washes (C). Proteins highlighted in yellow were selected for further analysis. (D) Venn diagram showing the numbers of ORFs (bold) or known translation factors (parentheses) identified in the three purification schemes.

Figure 3.

Novel proteins identified in the proteomic screens cosediment with ribosomes in an EDTA-dependent manner. Anti-TAP Western blots on sucrose gradient fractions in the absence (left) or presence (right) of EDTA. The locations of the ribosomal subunits in the gradients are indicated in the chromatograms and by Western blots of RPL3 and ASC1. CDC28 marks the nonribosomal fractions, and NOP1 shows the migration of a preribosomal complex

Figure 4.

Deletions of TMA proteins result in translation defects. (A) In vivo translation assay. Translation was measured as the amount of ³⁵S-methionine incorporation in yeast strains deleted for the indicated TMA protein relative to the isogenic wild-type (WT) strain. The standard deviations between three independent samples are shown. (*) A p-value of <0.05. (B) Nonsense suppression assay. Translation of a β-gal reporter gene containing either UAA, UAG, or UGA mutations, in deletion strains relative to the wild-type strain (set as 1). The standard deviations from at least three independent experiments with three distinct colonies for each strain are shown. (C) Resistance to translation inhibitors. YPD or YPD + 50 μg/mL anisomycin plates streaked with wild-type or Δtma7 after incubation for 2 d at 30°C are shown. (D) Polysome profile analysis. Absorbance at 254 nm of sucrose gradient fractions from wild type, Δtma19, Δtma7, and Δtma20. Arrows indicate differences in the mutant profiles compared with wild type.

Figure 5.

Human MCT-1 interacts with DRP1 and complements Δtma20 phenotypes. (A) α-DRP1 (top) or α-Flag (bottom) Western blots of cell lysates (Lys) and immunoprecipitates (IPs) from HEK293 cells transfected with Flag vector or Flag-MCT1. The lysate is 1/20 of the whole-cell extract used for the IP. (B) The amount of translation through nonsense mutations in a β-galactosidase reporter gene in wild-type (WT) and Δtma20 strains transfected with either empty expression vector (p413-GPD), yeast TMA20 (pTMA20), or the human MCT-1 gene expressed under control of the yeast GPD promoter (pGPD-MCT1). Error is reported as the standard deviation. (C) Polysome profiles from a wild-type yeast strain transfected with the empty pRS416 expression vector (left), the Δtma20 strain transfected with the empty pRS416 expression vector (middle), or Δtma20 transfected with an expression vector containing yeast TMA20 (pTMA20) (right). (D) Wild-type strain transfected with p416-GPD (left), Δtma20 strain transfected with p416-GPD (middle), or the Δtma20 strain transfected with pGPD-MCT1 (right). The arrow indicates the varying size of the 40S peak in the different profiles.