Mutations in the CMM2 domain affect MOM1 dimerization and MOM1-PIAL2 interaction.

<p><b>(A)</b> The L1761D/L1765D mutations in the CMM2 domain disrupted the CMM2 dimerization as determined by yeast two-hybrid assays. The wild-type CMM2 was fused to GAL4-BD; the wild-type and the mutated CMM2 were fused to GAL4-AD. “Vec” represents the empty <i>GAL-BD</i> or <i>GAL4-AD</i> vectors. <b>(B)</b> The full-length MOM1 harboring the CMM2 mutations still interacts with PIAL1 and PIAL2 in yeast. The wild-type and mutated MOM1 were fused to GAL4-BD; PIAL1 and PIAL2 were fused to GAL4-AD. “Vec” represents the empty <i>GAL-BD</i> or <i>GAL4-AD</i> vectors. <b>(C)</b> The mutations in the CMM2 domain disrupt MOM1 dimerization <i>in vivo</i> as determined by co-IP. The wild-type and mutated <i>MOM1-Flag</i> were introduced into transgenic plants harboring the <i>MOM1-Myc</i> transgene by crossing. F1 generation plants were subjected to co-IP. <b>(D)</b> The mutations in the CMM2 domain partially impair the interaction of MOM1 with PIAL2 <i>in vivo</i> as determined by co-IP. The wild-type and mutated <i>MOM1-Flag</i> were separately introduced into transgenic plants harboring <i>PIAL2-Myc</i> transgene by crossing. <b>(E)</b> The CMM2 mutations in the CMM2 domain of MOM1 are required for MOM1 complex formation <i>in vivo</i> as determined by gel filtration assays. Protein exaction from transgenic plants harboring the wild-type <i>MOM1-Flag</i> and the mutated <i>MOM1-CMM2-M-Flag</i> in the <i>mom1</i> background was eluted on Superose 6 (10/300 GL) column. The fractions were subjected to western blotting. The arrows mean the fractions corresponding to the standard proteins of 67, 220, 669 kDa.