Proteomic analysis of the 26S proteasome reveals its direct interaction with transit peptides of plastid protein precursors for their degradation.
The 26S proteasome is an ATP-dependent proteinase complex that is responsible for regulated proteolysis of polyubiquitinated proteins in eukaryotic cells. Here, we report novel 26S proteasome interacting proteins in Arabidopsis as revealed by LC-MS/MS analysis. We performed a two-step screening process that involved affinity purification of the 26S proteasome using Arabidopsis plants expressing a FLAG-tagged RPT2a subunit and partial purification of the 26S proteasome from cultured cells by glycerol density gradient centrifugation (GDG). Two plastid proteins, LTA2 and PDH E1α, which were commonly identified by both affinity purification and GDG, interacted with the 26S proteasome both in vitro and in vivo, and the transit peptides of LTA2 and PDH E1α were necessary for the interaction. Furthermore, the degradation of both LTA2 and PDH E1α was inhibited by MG132, a proteasome inhibitor. Similar to those two proteins, 26S proteasome subunits RPT2a/b and RPT5a interacted with the transit peptides of three other chloroplast proteins, which are known to be substrates of the ubiquitin-26S proteasome system. These results suggest that a direct interaction between the 26S proteasome and a transit peptide is important for the degradation of unimported plastid protein precursors to maintain cellular homeostasis.
Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Kaori
K
Sako
Yuki
Y
Yanagawa
Tomoyuki
T
Kanai
Takeo
T
Sato
Motoaki
M
Seki
Masayuki
M
Fujiwara
Yoichiro
Y
Fukao
Junji
J
Yamaguchi
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D060365:Chloroplast Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D036226:Seedlings / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease