Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

Abstract

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 multienzyme 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.

HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5′ UTR via a Newly Defined Repeat Motif in Embryophyta

“…Very bright GFP fluorescence perfectly overlapped with the autofluorescence of the chlorophyll ( Figure 3A), demonstrating the chloroplast localization of HCF145. In agreement with the previous data suggesting nucleoid localization of HCF145 (Phinney and Thelen, 2005), we also observed spots of GFP signal within the chloroplast. Even when the fluorescence microscope settings were set on the highest sensitivity, not even traces of GFP fluorescence were detected anywhere else within the cell, demonstrating that HCF145 is very likely exclusively localized in the chloroplast ( Figure 3A).…”

“…In a large-scale proteome approach, peptides of HCF145 were found in triton-insoluble fractions, representing the nucleoid fraction of the chloroplast (Phinney and Thelen, 2005).…”

New insights into plastid nucleoid structure and functionality

“…In the TAC proteome analysis described by Pfalz et al (2006) a PPR protein with a molecular weight of 97 kDa (pTAC2) was identified. Interestingly, Phinney and Thelen (2005) identified a pea protein of similar size in their nucleoid analysis which matched the monoisotopic masses of the same PPR protein, corroborating its presence in chloroplast nucleoids. In a recent analysis of spinach TAC, two further PPR proteins of 98 and 67 kDa were identified (Melonek et al 2012).…”

“…The TAC fraction is prepared from plastid membranes by release of the nucleoids with detergents and by subsequent purification involving sepharose gel filtration and other steps (Krause and Krupinska 2000;Pfalz et al 2006). Recent proteomic analyses of these (Pfalz et al 2006;Melonek et al 2012) and other nucleoid-enriched fractions (Phinney and Thelen 2005;Majeran et al 2012) have revealed that several of the newly identified DNA binding proteins were not inherited from the prokaryotic ancestors. Plastid proteins homologous to the abundant prokaryotic HU protein, on the other hand, were not detected in higher plants which stands in contrast to their identification in some algal plastids (Karcher et al 2009), in the apicoplasts of apicomplexans (Sato et al 2003) as well as in some dinoflagellates (Chan et al 2006).…”

“…Accordingly, core subunits of the plastid-encoded RNA polymerase, PEP, have been identified in most of the proteomic studies of plastid nucleoid fractions of dicots (i.e. Arabidopsis, mustard, spinach, pea) (Phinney and Thelen 2005;Pfalz et al 2006;Melonek et al 2012) as well as monocots (i.e., maize) (Majeran et al 2012). A preparation, where only the core PEP and very tightly associated subunits were preserved, contained an additional ten proteins that were termed PEP-associated proteins (for PAPs) (Steiner et al 2011).…”