PMC:2728203 / 8258-10182 JSONTXT

{"project":"2_test","denotations":[{"id":"18190927-12531921-62517981","span":{"begin":13,"end":15},"obj":"12531921"},{"id":"18190927-12496292-62517982","span":{"begin":242,"end":244},"obj":"12496292"},{"id":"18190927-11403570-62517983","span":{"begin":471,"end":473},"obj":"11403570"},{"id":"18190927-15208322-62517984","span":{"begin":1562,"end":1564},"obj":"15208322"}],"text":"Previous work21 showed that full-length Nab2 binds to the C-terminal globular domain of Mlp1 (CT-Mlp1). We utilized the yeast two-hybrid system to identify the domain of Nab2 that interacts with CT-Mlp1. Nab2 can be divided into four domains,23 and hence, we constructed a series of two-hybrid bait plasmids expressing Nab2 in which each of the individual domains had been deleted (Fig. 1a). We used a lacZ reporter that generates a blue color when an interaction occurs.29 Each Nab2 plasmid was coexpressed with the vector alone (pJG4–5) or CT-Mlp1 (Fig. 1a). As shown in Fig. 1a, the negative vector controls (pEG202 and pJG4–5) were white, confirming that they did not activate the lacZ reporter. In contrast, coexpression of CT-Mlp1 and Nab2 generated the blue color, indicating that the lacZ reporter was activated, consistent with the proteins interacting with one another. CT-Mlp1 failed to interact with Nab2 only when the N-terminus was deleted (ΔN-Nab2), and the interaction was maintained with all other Nab2 fusion proteins. To determine if the N-terminal domain of Nab2 was sufficient for interaction with CT-Mlp1, we attempted to create a minimal yeast two-hybrid construct expressing only the N-terminal domain of Nab2. Unfortunately, this N-terminal domain fusion protein autoactivated the lacZ reporter; thus, we instead created a fusion consisting of both the N-terminal domain and the QQQP domain (Nab2-NQ). This protein interacted with CT-Mlp1 (Fig. 1a). Gfd1 was used as a positive control for interaction with the N-terminal domain of Nab2,27 and, as expected, Gfd1 interacted with the NQ domain but not with ΔN-Nab2 (Fig. 1a). For all two-hybrid experiments, expression of each fusion protein was confirmed by immunoblotting (data not shown). Overall, the two-hybrid data suggest strongly that the N-terminal domain of Nab2 is both necessary and sufficient to interact with the Mlp1 C-terminal domain."}