Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex.
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 (1M 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.
Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire 03824, USA.
Xin
X
Wang
Chongxu
C
Zhang
Yueh-Chin
YC
Chiang
Shaun
S
Toomey
Matthew P
MP
Power
Mitchell E
ME
Granoff
Roy
R
Richardson
Wen
W
Xi
Darren J
DJ
Lee
Susan
S
Chase
Thomas M
TM
Laue
Clyde L
CL
Denis
D039561:Eukaryotic Initiation Factor-4E / Q000302:isolation & purification / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000302:isolation & purification / Q000378:metabolism; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000302:isolation & purification / Q000378:metabolism; D054657:Ribosome Subunits / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods*
D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039102:Poly(A)-Binding Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C450100:pab1 protein, S cerevisiae; D011189:Potassium Chloride; D005947:Glucose