2009-04-25

62# Fleisher et al. 2006Gene Dev.-gradient de Ypl009c

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 !tma20 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.

First, 40S, 60S, 80S, and polyribosomal complexes were fractioned using sucrose gradients. Second, ribosomes were purified under increasing salt concentrations using discontinuous sucrose gradients. Third, ribosome salt washes(RSW) with three different salt concentrations were used to dissociate potential regulatory factors from core ribosomes.