Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.
In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.
Baginsky
S
S
Institute of Plant Sciences, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
Shteiman-Kotler
A
A
Liveanu
V
V
Yehudai-Resheff
S
S
Bellaoui
M
M
Settlage
R E
RE
Shabanowitz
J
J
Hunt
D F
DF
Schuster
G
G
Gruissem
W
W
D018834:Chaperonin 60 / Q000737:chemistry; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D010788:Photosynthesis; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D018724:Spinacia oleracea
D018834:Chaperonin 60; D009097:Multienzyme Complexes; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; D020365:RNA Helicases