PMC:2728203 / 8217-12449 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"The Nab2 N-terminal domain binds to Mlp1\nPrevious 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.\nPull-down assays complemented the results obtained using the two-hybrid system and confirmed that the N-terminal domain of Nab2 without the QQQP domain is both necessary and sufficient to mediate interactions with Mlp1. A glutathione S-transferase (GST) fusion to the N-terminal domain of Nab2 (GST-Nab2-N, residues 1–97 of Nab2) was engineered, expressed in Escherichia coli, and purified. Either GST-Nab2-N or GST alone as a control protein was incubated in yeast lysate, and then, the GST protein and any associated proteins were bound to glutathione beads. As shown in Fig. 1b, a band of approximately 220 kDa that copurified with GST-Nab2-N but not with GST alone was visualized using Coomassie Blue staining. Immunoblotting confirmed that this band corresponded to the full-length Mlp1 protein (Fig. 1c). When the yeast lysate was prepared from mlp1Δ cells, this 220-kDa band was not detected by either Coomassie staining (Fig. 1b) or immunoblotting (Fig. 1c). Although the amount of Mlp1 copurified with GST-Nab2-N from yeast lysate was rather small, it is quite remarkable that a band corresponding to this full-length, very large, nuclear pore-associated protein17 can be isolated from yeast extract in a single step. The results of this experiment indicate that the N-terminal domain of Nab2 is sufficient to copurify Mlp1 from a complex mixture, showing that the interaction is highly specific.\nAn in vitro binding assay established that the N-terminal domain of Nab2 interacted directly with the Mlp1 C-terminus. Recombinant proteins were expressed and purified from E. coli as described in Materials and Methods. Either GST-Nab2-N or GST control protein was incubated with the Mlp1 C-terminal domain fragment. As shown in Fig. 1d, the Mlp1 fragment bound to GST-Nab2-N but not to the control GST protein, confirming that the N-terminal domain of Nab2 interacts directly with the C-terminal domain of Mlp1. The amount of binding between Nab2-N and CT-Mlp1 appears to be substoichiometric, which is consistent with the interaction between Nab2 and Mlp1 being relatively weak, to facilitate export rather than retention at the nuclear pore. Preliminary binding studies indicated that the Kd for this interaction is in the micromolar range (data not shown).","divisions":[{"label":"title","span":{"begin":0,"end":40}},{"label":"p","span":{"begin":41,"end":1965}},{"label":"p","span":{"begin":1966,"end":3371}}],"tracks":[{"project":"2_test","denotations":[{"id":"18190927-12531921-62517981","span":{"begin":54,"end":56},"obj":"12531921"},{"id":"18190927-12496292-62517982","span":{"begin":283,"end":285},"obj":"12496292"},{"id":"18190927-11403570-62517983","span":{"begin":512,"end":514},"obj":"11403570"},{"id":"18190927-15208322-62517984","span":{"begin":1603,"end":1605},"obj":"15208322"},{"id":"18190927-10085285-62517985","span":{"begin":3137,"end":3139},"obj":"10085285"}],"attributes":[{"subj":"18190927-12531921-62517981","pred":"source","obj":"2_test"},{"subj":"18190927-12496292-62517982","pred":"source","obj":"2_test"},{"subj":"18190927-11403570-62517983","pred":"source","obj":"2_test"},{"subj":"18190927-15208322-62517984","pred":"source","obj":"2_test"},{"subj":"18190927-10085285-62517985","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecaa93","default":true}]}]}}