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Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments

Permanent URL:
http://handle.nal.usda.gov/10113/54539
Abstract:
The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state 13C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U‐13C]‐sucrose, [U‐13C]‐glucose, [U‐13C]‐glutamine or [U‐13C]‐alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography‐mass spectrometry. Amino acids including alanine, glycine and serine exhibited different 13C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed.
Author(s):
DOUG K. ALLEN , RUSSELL W. LACLAIR , JOHN B. OHLROGGE , YAIR SHACHAR‐HILL
Subject(s):
Brassica napus , alanine , amino acid metabolism , biosynthesis , cytosol , embryo culture , enzymatic reactions , eukaryotic cells , glycine (amino acid) , isotope labeling , labeling , organelles , plant architecture , proteins , ribulose , ribulose-bisphosphate carboxylase , serine
Source:
Plant, cell & environment 2012 7 v.35 no.7
Language:
English
Publisher:
Blackwell Publishing Ltd
Year:
2012
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
File:
Download [PDF File]
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.