Self-association of mr-s protein
SAM domains are known to function as protein-protein interaction modules [15-17]. Although SAM domains can bind to various non-SAM domain-containing proteins, many homo-SAM and hetero-SAM domain interactions have been reported. To investigate whether the SAM domain of the mr-s protein can also function as a protein-protein interaction module, we performed yeast two-hybrid screening using full-length mr-s protein as the bait. Using this bait, we screened the transcriptional activator fusion protein library in which mouse P0-P3 retinal cDNAs were fused to the GAL4 activation domain. The most frequent positive clones (5 out of 28) were cDNA fragments containing the SAM domain of mr-s (Fig. 4A). This result strongly suggests that mr-s protein self-associates through SAM domain-containing regions. We then directly tested this self-association of mr-s protein in yeast. We fused full-length or truncated portions of the mr-s protein to the DNA-binding domain of the yeast transcription factor GAL4 to make bait constructs. We fused full-length or truncated portions of the mr-s protein to the GAL4 transcriptional activation domain to make prey constructs (Fig. 4B). These constructs were transformed into yeast that contain a transgene with GAL4 binding sites upstream of the lacZ gene. We found that the full-length mr-s bait construct induced lacZ expression with the full-length mr-s prey construct (Fig. 4B, full × full). The N-terminus 400 amino acid (aa) stretch of mr-s, which does not contain a SAM domain, does weakly activate transcription of lacZ (Fig. 4B, full × N). The N-terminus 400 aa stretch of mr-s was able to induce transcription of lacZ weakly with the same N-terminus 400 aa stretch of mr-s (Fig. 4B, N × N). Although the N-terminus 400 aa mr-s protein weakly activates lacZ transcription with the same N-terminus portion, a much stronger activation of lacZ expression was observed with a C-terminus portion encoding the 391–542 aa stretch of mr-s (Fig. 4B, full × C, C × C). Our GAL4 assay indicated that the signal when the full-length mr-s was present in both the bait and prey contexts was weaker than when isolated SAM domains were used. This may simply reflect the tendency for the small fusion proteins to enter the yeast nucleus and occupy GAL4 binding sites. Alternatively, the SAM domain may be less accessible for interaction in the full-length protein context as previously reported [39].
Figure 4  Summary of yeast two-hybrid screening and GAL4 assay. (A) Full-length mr-s as a bait used in the yeast two-hybrid screening and positive clones are indicated. Note that all of five mr-s clones identified in the screening contain SAM domains. (B) Schematic diagram of the mr-s fusion proteins used in the yeast GAL4 assay. Black boxes represent the position of SAM domains. Relative levels of LacZ expression are shown on the right, respectively. Note; ++ indicates an intense blue signal visible after 12hr of incubation at 37°C, + indicates a blue signal visible after 24hr of incubation. BD, binding domain; AD, activation domain; full, full-length mr-s; N, N-terminal portion of mr-s (amino acids 1 to 400); C, C-terminal portion of mr-s (amino acids 391 to 542). To confirm self-association of the mr-s protein in mammalian cells, we next performed co-immunoprecipitation studies in HEK293T cells by co-transfection of HA-tagged full-length/truncated mr-s and Flag-tagged full-length/truncated mr-s (Fig. 5A). As a negative control, we constructed Flag-tagged Sonic hedgehog (Shh) (lane 2 and 7). In accordance with the result of the yeast two-hybrid GAL4 assay, HA-tagged full-length mr-s (full-HA) was co-immunoprecipitated with Flag-tagged full-length mr-s (Flag-mrs) and the Flag-tagged C-terminus portion containing the SAM domain (Flag-SAM), respectively (Fig 5B, lane3 and lane5). We also found a weak co-immunoprecipitation band in co-transfection of full-HA and Flag-tagged N-terminus portion of mr-s (Flag-ΔSAM, Fig. 5B, lane 4). When ΔSAM-HA and Flag-tagged deletion mutants were co-transfected, ΔSAM-HA was co-immunoprecipitated with Flag-mrs and Flag-ΔSAM (Fig. 5B, lane 8 and lane 9), while ΔSAM-HA was not co-immunoprecipitated with Flag-SAM (Fig. 5B, lane 10).
Figure 5  The mr-s protein can self-associate in mammalian cells. (A) Schematic drawing of the constructs used for immunoprecipitation assay. HA-tagged or Flag-tagged full-length (amino acids 1 to 542), ΔSAM (amino acids 1 to 400) and SAM (amino acids 401 to 542) regions were inserted into pcDNA3 vector, respectively. (B) The constructs indicated above were transfected into HEK293T cells. Each lane was co-immunoprecipitated by anti-Flag antibody and detected by anti-HA antibody. Input protein lysates are shown in the lower panels. (C) Flag-tagged two site-directed mr-s mutants, Flag-W404A and Flag-G453A were generated and co-transfected with full-HA. Each lane was co-immunoprecipitated by anti-HA antibody and detected by anti-Flag antibody. To investigate whether the mr-s protein self-associates mainly through the SAM domain, two site-directed mutations were generated in the SAM domain of mr-s (Fig. 1B, arrows). These mutations alter residues that are conserved in the SAM domain of ph and previous report indicates that these mutations of ph-SAM cause significant reduction in binding activity to the other SAM domain-containing protein, Sex comb on midleg (Scm) (41). Based on this result, we introduced two types of site-directed mutations, which correspond to the mutations introduced in ph protein, into Flag-tagged full-length mr-s (Flag-W404A and Flag-G453A). We found that Flag-W404A binding activity was significantly reduced and Flag-G453A binding activity was also slightly reduced compared to Flag-mrs (Fig. 5C). These results, together with yeast two-hybrid GAL4 assay, indicate that the mr-s protein self-associates strongly through its SAM domain and weakly through the N-terminus portion lacking SAM domain.