Figure 6.

WKS1.1 Chloroplasts Import Assays.

(A) Radiolabeled full-length WKS1.1 protein incubated with intact chloroplasts. tl = 10% of translation products used for the import assay; imp. = proteins in intact chloroplasts recovered after a 20-min import reaction; Na₂CO₃ = chloroplasts lysed with 0.1 M Na₂CO₃ and fractionated by centrifugation into a supernatant that contained soluble proteins (S) and a pellet that contained integral membrane proteins (IM); thermolysin = chloroplasts incubated on ice for 30 min without (−) or with (+) thermolysin, or with thermolysin and 2% (v/v) Triton X-100 (Tx). Radiolabeled proteins were visualized using a phosphor imager: FL; full-length WKS1.1 (72 kD); M, processed M-WKS1.1 (66 kD).

(B) Radiolabeled full-length WKS1.1 protein incubated with intact chloroplasts for the time indicated at the top of the gel and analyzed as described in the legend of (A). Intensity of each band relative to the total WKS1.1 protein (FL+M) is indicated above the bands.

(C) and (D) Radiolabeled WKS1.1-GFP fusion protein (C) and radiolabeled START domain protein (D) incubated with intact chloroplasts and analyzed as described in the legend of (A).

The sizes of molecular mass markers in kilodaltons are shown to the left of each image. Import of control proteins DGD1 (located at the outer membrane) and Tic40 (located at the inner membrane) can be found in Supplemental Figure 8.