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. 2015 Jan 13;27(1):262–285. doi: 10.1105/tpc.114.132787

Figure 10.

Figure 10.

Interaction of MET1 with Thylakoid Proteins Determined by Co-IP and Y2H Analysis.

(A) Co-IP of MET1 with anti-MET1 serum against DM solubilized thylakoids to identify potential protein interactors. Immunoblotting with various specific antisera showed that MET1 was highly enriched in the co-IP and that both CP43 and CP47 interact with MET1. Thylakoids from the met1-1 mutant were used as negative control.

(B) Split-ubiquitin assays for interactions between full-length MET1 and selected thylakoid proteins. Full-length MET1 was used as bait and selected candidate proteins were used as prey. Bait plasmid contains Cub-PLV and prey plasmid contains NubG. NubG moiety was fused to the N terminus of prey proteins. The resulting plasmids were transformed into the yeast bait and prey strains. The transformed yeast strains harboring bait and prey constructs were mated and resulting transformants were analyzed on selective medium lacking Ade, His, Trp, Leu, Ura, and Met (upper lane) and for β-galactosidase (β-Gal) activity (lower lane). Soluble NubG and Nub-WT were used as negative and positive controls, respectively.

(C) Interaction of the TPR domain of MET1 with CP43 and CP47. Diploid cells were analyzed on selective medium lacking Ade, His, Trp, Leu, Ura, and Met (upper lane) and for β-Gal activity (lower lane).

(D) Interaction of PDZ domain of MET1 with CP43 and CP47. Diploid cells were analyzed on selective medium lacking Ade, His, Trp, Leu, Ura, and Met (upper lane) and for β-Gal activity (lower lane).

(E) and (F) Interactions between full-length MET1 and stromal loops (B, D, and C-terminal) and lumenal loop E of CP43 (E) and CP47 (F). Diploid cells were analyzed on selective medium lacking Ade, His, Trp, Leu, Ura, and Met (upper lane) and for β-Gal activity (lower lane).

(G) Enrichment analysis of RNCs extracted from thylakoids of the wild type. ALB3 but not MET1 is highly enriched in such RNC preparations. Solubilized thylakoid proteins of the wild type and met1-1 were used for reference of the immunoblots.