Figure 1

Identification of a novel Mid1 partner. (A) Interaction-mating assay that confirms Mid1-Mig12 interaction in yeast. B42 fl, Mig12 full-length fused to the B42 activation domain; B42 or, the largest original Mig12 clone fused to the B42 activation domain; LexA Mid, constructs encompassing different MID1 domains fused to the LexA DNA binding domain: A, full-length; C, BB; D, CC; F, RFP-like; H, R-BB-CC; M, CC-FNIII-RFP-like. Both the full-length and the original Mig12 clones specifically interact with the entire Mid1 protein and with some of its truncated mutants, MidD, MidH and MidM, as shown by yeast turning blue on X-gal plates and growing on plates lacking leucine (Leu), only when galactose (Gal), and not glucose (Glu), is used as carbon source. Abbreviations: BB, B-box1 and B-box2 domains; CC, coiled-coil domain; FNIII, fibronectin type III repeat; R, RING domain. (B) Amino acid sequence of human (h) and mouse (m) MIG12 and comparison with the zebrafish G12 and the human SPOT14 proteins. Amino acids that are identical at least in the human and murine Mig12 are in bold. Conserved amino acids are indicated in gray. The human and mouse MIG12 share 90% of similarity and 88% of identity. The hMIG12 and the zebrafish protein share 56% of similarity and 46% of identity, whereas the homology with the human SPOT-14 protein is 49% and 31%, respectively. There is a gap of 25 aa that are not present in the zebrafish and SPOT14 proteins. (C) Co-immunoprecipitation experiments showing Mid1-Mig12 interaction. Western blot (WB) analysis using anti-Mid1 and anti-HA antibodies after immunoprecipitation of HEK293 cells transiently transfected with different combination of MycGFP-tagged Mid1 (MGFP-MID1) and an HA-tagged Mig12 (HA-MIG12); + and - indicate the constructs transfected in each lane. The antibodies used for the immunoprecipitations (IP) are indicated. Mid1 indicates the band corresponding to the endogenous protein. Ig, immunoglobulins. In some experiments, we detected a trace amount of MGFP-Mid1 immunoreactivity in cells transformed with only MGFP-Mid1 and immunoprecipitated with the anti-HA antibody. This signal was always much less than that seen when both tagged constructs were transfected together. (D) The same as in (C) using the MGFP-MidM, MGFP-MidH and MGFP-MidD mutant fusions, instead of the full-length protein, in the co-transfections and an anti-Myc antibody for Western blot analysis.