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Mapping of Ndrg2 and MSP58 interacting domain in yeast
To map the region(s) of Ndrg2 and MSP58 involving their interaction, various truncated mutants of Ndrg2 and MSP58 were subjected to the analysis in yeast two-hybrid assay. Except for the full-length of Ndrg2, any truncation of Ndrg2 could completely abolish its interaction with MSP58 (data not shown). These results implicated the integrity of Ndrg2 protein is necessary for stable interaction with MSP58. The N-terminal-deleted MSP58 SCH900776 (MSP58115–462, MSP58194–462, and MSP58274–462) are keeping the interaction ability with Ndrg2. In contrast, the C-terminal truncated MSP58 proteins (MSP58115–361 and MSP58115–310) failed to interact with Ndrg2 (Fig. 1A). The results implicated the C-terminal region of MSP58 is necessary for its interaction with Ndrg2.
Ndrg2 interacts with MSP58 directly in vitro
Next, we tested whether the direct association exists between Ndrg2 and MSP58 by using GST pull-down assay. GST-MSP58 fusion protein or GST fusions with the deletion mutants of MSP58 were incubated with the COS7 cell lysate containing Myc-tagged Ndrg2 proteins produced by pCMV-Myc-NDRG2 transfection. The Ndrg2 binding to the various GST fusions was detected by anti-Myc antibody blotting. As shown in Supplement 1and Fig. 1B, Ndrg2 protein could be specifically pulled down by GST-MSP58 fusion protein but not by GST alone.