Proteomic characterization of a triton-insoluble fraction from chloroplasts defines a novel group of proteins associated with macromolecular structures.
Proteomic analysis of a Triton X-100 insoluble, 30,000 x g pellet from purified pea chloroplasts resulted in the identification of 179 nonredundant proteins. This chloroplast fraction was mostly depleted of chloroplast membranes since only 23% and 9% of the identified proteins were also observed in envelope and thylakoid membranes, respectively. One of the most abundant proteins in this fraction was sulfite reductase, a dual function protein previously shown to act as a plastid DNA condensing protein. Approximately 35 other proteins known (or predicted) to be associated with high-density protein-nucleic acid particles (nucleoids) were also identified including a family of DNA gyrases, as well as proteins involved in plastid transcription and translation. Although nucleoids appeared to be the predominant component of 30k x g Triton-insoluble chloroplast preparations, multi-enzyme protein complexes were also present including each subunit to the pyruvate dehydrogenase and acetyl-CoA carboxylase multi-enzyme complexes, as well as a proposed assembly of the first three enzymes of the Calvin cycle. Approximately 18% of the proteins identified were annonated as unknown or hypothetical proteins and another 20% contained "putative" or "like" in the identifier tag. This is the first proteomic characterization of a membrane-depleted, high-density fraction from plastids and demonstrates the utility of this simple procedure to isolate intact macromolecular structures from purified organelles for analysis of protein-protein and protein-nucleic acid interactions.
Phinney
Brett S
BS
Michigan State University, Proteomics and Mass Spectrometry Facility, East Lansing, Michigan 48824, USA.
Thelen
Jay J
JJ
D015153:Blotting, Western; D002736:Chloroplasts / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D014176:Protein Biosynthesis; D020543:Proteome; D014158:Transcription, Genetic
D010940:Plant Proteins; D020543:Proteome; D011092:Polyethylene Glycols