Table 2.
Preliminary analysis of a selection of chloroplast-associated polypeptides, following biotinylation or thermolysin treatment and 2D-PAGE resolution.
| Polypeptides associated to the spinach envelope membranes based on immunostaining (*), MS- (**), or MS/MS (***) analyses | Uniprot reference | Spot number | Mr (kDa) | Isoelectric point | Tag and shave | Protein localization (in Arabidopsis) | |||
|---|---|---|---|---|---|---|---|---|---|
| Match | Gel | Match | Gel | Biotinylation | Thermolysin sensitivity | ||||
| STROMAL RNA EDITING | |||||||||
| 28RNP 28 kDa ribonucleoprotein (***) | P28644a | 72 | 25.2 | 35.0 | 4.40 | 4.50 | − | +++ | Stroma1 + IEM1 |
| CSP41 mRNA-binding protein (***) | O24365a | 50 | 44.8 | 46.0 | 6.11 | 6.00 | – | – | Stroma1 + Thyl1 + IEM1 |
| PROTEIN IMPORT COMPONENTS, CHAPERONES, AND PROTEASES | |||||||||
| Cytosolic HSP70/Com70 (***) | P29357a | 7 | 71.7 | 74.0 | 5.12 | 5.20 | ++ | +++ | Cytosol2 + OEM2 |
| Translocon Tic110 (***) | Q8LPR9b | 1 | 109.9 | 110.0 | 5.21 | 5,20 | – (+ upper part of spot 1) | – (+ upper part of spot 1) | IEM1 |
| Translocon Tic40-like protein (***) | Q9FMD5b | 25 | 49.2 | 50.0 | 5.38 | 4.50 | – | +++ | IEM1 |
| ClpC, member of the HSP100 family (**) | O98447a | 2, 3, 4 | 99.4 | 99.0 | 8.78 | 5.50 | – | +++ | IEM1 + Stroma1 |
| ClpP5, subunit of the ATP-dependent Clp protease (**) | Q9S834b | 62 | 32.3 | 30.0 | 8.35 | 6.50 | – | – | Stroma1 + IEM1 |
| HSP70, member of the DNAK family (***) | O50036a | 9 | 76.1 | 74.0 | 5.19 | 4.50 | + | +++ | Stroma1 |
| Cpn21, member of the GroEL family, co-chaperone of Cpn60 (***) | Q02073a | 60 | 26.9 | 35.0 | 8.45 | 6.50 | – | – | Stroma1 + IEM1 |
| Cpn60β, member of the GroES family, Rubisco binding-protein (***) | P08927c | 17 | 62.9 | 60.0 | 5.85 | 5.30 | – (+ on part of spot 84) | – | Stroma1 |
| RUBISCO, RUBISCO MATURATION AND ASSEMBLY | |||||||||
| Rubisco LSU (**) | P00875a | 23, 24 | 52.7 | 53.0 | 6.13 | 6.40 | – (+ upper part of spot 23-24) | – (+ upper part spot 23-24) | Stroma1 + Env1 |
| Rubisco SSU 2 (**) | Q43832a | 84 | 20.3 | 21.0 | 8.24 | 6.50 | – | – | Stroma1 + Env1 |
| Rubisco SSU N-methyltransferase I (***) | Q9TIM3a | 10 | 54.9 | 57.0 | 5.16 | 4.50 | – | – | Stroma1 |
| Rubisco activase (**) | P10871a | 30, 38, 40 | 51.5 | 48–52.0 | 6.62 | 5.30 | – | – | Stroma1 |
| OTHER CARBON METABOLISM COMPONENTS | |||||||||
| Hexokinase 1 (***) | Q9SEK3a | 20, 21 | 54.1 | 60.0 | 5.41 | 5.80 | +++ | +++ | OEM1 + Mit./Vac./Nucl.3 |
| Carbonic anhydrase (***) | P16016a | 60 | 34.5 | 34.0 | 6.61 | 6.30 | – | – | IEM1 + Stroma1 |
| Phosphoglycerate kinase (***) | P29409a | 54 | 45.5 | 42.0 | 5.83 | 6.50 | – | – | Stroma1 |
| Fructose-1,6-biphosphate aldolase (***) | P16096a/ P29356a | 49 | 42.4 | 44.0 | 6.85 | 5.80 | + | – | Stroma1/Cytosol3 |
| Fructose-1,6-biphosphatase (***) | P22418a | 55 | 37.1 | 37.0 | 5.52 | 5.50 | + | – | Stroma1 |
| Sedoheptulose-1,7-bisphosphatase (***) | O20252a | 28 | 42.0 | 45.0 | 5.87 | 5.00 | – | – | Stroma1 |
| Transketolase (**) | O20250a | 5, 6 | 80.2 | 75.0 | 6.20 | 5.50 | – | – | Stroma1 |
| Phosphoribulokinase (***) | P09559a | 39 | 44.9 | 49 | 5.82 | 5.70 | + | – | Stroma1 |
| NAD-malate dehydrogenase (***) | O81609c | 47 | 42.6 | 35.0 | 7.01 | 5.5 | – | – | IEM1 + Stroma1 |
| THYLAKOID NUCLEAR ENCODED CF-1 SUBUNITS | |||||||||
| CF1-ATP synthase alpha chain (**) | P06450a | 11 | 55.4 | 57.0 | 5.16 | 5.20 | – | – | Thyl1 + Env1 |
| CF1-ATP synthase beta chain (**) | P00825a | 16, 18 | 53.7 | 54.0 | 5.22 | 5.55 | – | – | Thyl1 |
| CF1-ATP synthase delta chain (**) | P11402a | 74 | 27.6 | 28.0 | 5.80 | 4.50 | – | – | Thyl1 + Env1 |
| ENVELOPE GLYCEROLIPID AND QUINONE METABOLISMS | |||||||||
| MGD1 monogalactosyldiacylglycerol synthase (*) | Q9SM44a | 12–15 | 53.7 | 54.0 | 5.22 | 5.00 | – | – | IEM1 |
| IEP37 quinone methyltransferase (*) | P23525a | ~26 | 36.8 | 36.0 | 9.49 | 4–7 | – | – | IEM1 |
| CeQORH quinone oxydoreductase homolog (*) | Q9SV68b | nd | 34.4 | 40.0 | 9.05 | 9.60 | – | – | IEM1 |
| PLASTID DIVISION | |||||||||
| FtsZ2 (*) | O82533b | nd | 45.2 | 50.0 | 5.01 | 6.00 | – | +++ | IEM4 + Stroma1,3 + Thyl3 |
| OTHER ENVELOPE PROTEINS | |||||||||
| OEP24 (*) | Q41393a | 73 | 16.2 | 29.0 | 4.84 | 4.80 | +++ | +++ | OEM1 |
| OEP10 (* and ***) | P19407a | 8 | 6.4 | 110.0 | 6.01 | 4.5 | +++ | +++ | OEM1 |
The tag and shave intensity is given as a scale: −, no effect on the characterized spot; +, ++, +++, increasing effects of the tag or shave treatment. For biotinylation, the scale is estimated by the average size of the biotinylated spots being smaller, +; identical, ++; or bigger, +++; when compared to the size of the overlapping Coomassie-stained spots. (+), partial tag or shave of a large 2-D PAGE spot. UniProt accession numbers were given from
Spinacea oleracea;
Arabidopsis thaliana; and
Pisum sativum. Abbreviations: Env, envelope; IEM, inner envelope membrane; Mit, mitochondria; OEM, outer envelope membrane; Thyl, thylakoïd; nd, not defined after Coomassie staining; Nucl, nucleus; Vac, vacuole. Previoulsy determined localization of proteins was obtained from works by
Ferro et al. (2010),
Ko et al. (1992),
Heazlewood et al. (2007), and
El-Kafafi et al. (2005).