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Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex
Author(s) -
Wang Xin,
Zhang Chongxu,
Chiang YuehChin,
Toomey Shaun,
Power Matthew P.,
Granoff Mitchell E.,
Richardson Roy,
Xi Wen,
Lee Darren J.,
Chase Susan,
Laue Thomas M.,
Denis Clyde L.
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2110
Subject(s) - eif4e , eif4g , stress granule , initiation factor , translational regulation , poly(a) binding protein , translation (biology) , chemistry , eukaryotic translation , ribosome , saccharomyces cerevisiae , protein biosynthesis , biochemistry , biology , messenger rna , biophysics , yeast , rna , gene
A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU‐FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae . Following a one‐step affinity purification of either poly(A)‐binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP‐tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed‐loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1 M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.