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 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 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 ªputativeº or ªlikeº 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