THA8 partitions between the chloroplast stroma and the extrinsic face of thylakoid membranes. (A) THA8 is enriched in isolated chloroplasts. Immunoblots of leaf, chloroplast (cp), and mitochondrial (mito) extracts from wild-type (WT) and tha8 mutant seedlings probed with antibodies to THA8 or the mitochondrial protein malate dehydrogenase (MDH). A portion of each Ponceau S-stained membrane is shown to illustrate sample loading. (B) Intra-chloroplast location of THA8. Purified chloroplasts were separated into stromal and thylakoid fractions. The thylakoids were washed with Na2CO3 to remove extrinsic proteins, repelleted (P) and the supernatant (S) removed. Equivalent proportions of each fraction were analyzed by immunoblotting with antibodies to THA8 and AtpA, a membrane-extrinsic subunit of the ATP synthase. Unfractionated tha8 and WT chloroplasts are shown to the left.
