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Fig. 6. VAR3 protein interaction and targeting. (A) Yeast two-hybrid interactions. Left panel shows growth of yeast strains carrying different plasmids on minimal medium lacking Trp, Leu, His and adenine. Right panel identifies fusion proteins expressed from the DNA-binding (BD bait) and activation domain (AD prey) plasmids in the yeast strains shown on the left. LAM is human lamin C, TD1 and VA3 (B) Co-immunoprecipitation (Co-IP) of VAR3 and NCED4. (Left to right) Lane 1, IP of cMyc-tagged VAR3; lane 2, IP of HA-tagged NCED4 (partial length open reading frame isolated from the cDNA library screen); lane 3, IP of HA-tagged LAM; lane 4, Co-IP of NCED4 with cMyc-tagged VAR3; lane 5, lack of Co-IP of control LAM with cMyc-tagged VAR3. Proteins and antibodies are noted above. (C) In vitro chloroplast import assay. Isolated, intact chloroplasts were incubated with in vitro translated, radiolabeled precursor proteins, and chloroplasts were then fractionated and samples analyzed by SDS-PAGE and fluorography. Gel lanes are: Tr, in vitro translation mixture; Tr+, translation mixture digested with thermolysin; C, total chloroplasts; C+, thermolysin-treated chloroplasts; S, stroma; T, thylakoid membranes; T+, thermolysin-treated thylakoids. Pre- and m- denote precursor and mature protein, respectively. SSU is a RbcS stromal marker and 33 K is the 33 kDa subunit of the oxygen-evolving complex, thylakoid lumen marker. The sizes (kDa) of precursor and mature forms of VAR3 and NCED4 are indicated.

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