Biophysical properties of the eukaryotic ribosomal stalk.
The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.
Grela
Przemysław
P
Department of Molecular Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.
Krokowski
Dawid
D
Gordiyenko
Yuliya
Y
Krowarsch
Daniel
D
Robinson
Carol V
CV
Otlewski
Jacek
J
Grankowski
Nikodem
N
Tchórzewski
Marek
M
D055592:Biophysical Phenomena / Q000235:genetics; D049109:Cell Proliferation; D056890:Eukaryota / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics; D005818:Genetic Engineering; D011499:Protein Processing, Post-Translational; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C056003:RPP1A protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C054675:ribosomal protein P0; D020558:GTP Phosphohydrolases