Remorin 6.6, a membrane nanodoamin protein, interacts with myosin VIII and microtubules in a phosphorylation dependent manner and affects plant growth

Remorins (REM) are proteins associated with membrane nanodomains and there are indications for their involvement in stress response. The family of Arabidopsis remorins includes 16 members. Class VIII myosins are also associated with the plasma membrane, and Pearson’s coefficient analysis of colocalization data between 4 myosin VIII, and 11 remorin family members revealed the colocalization of ATM1 with REM6.6 in N. benthamiana (N.b). Serial deletions revealed that the N-terminus of REM6.6, was accumulated in the cytoplasm, however, in the presence of ATM1, it was recruited to the plasma membrane, suggesting a physical interaction between the two proteins, which was confirmed also by FRET analysis. Interestingly, REM6.6 is also detected along microtubules (MTs) in both N.b and Arabidopsis plants, an association which is enhanced by stress signals or ectopic application of ABA. In N.b, less REM6.6 was detected along MTs after ABA application if ATM1 was co-expressed, suggesting a competitive interaction. Six phosphorylation sites were found in the N-term of REM6.6 by the analysis of phosphopeptides; Ser 38, 108, 127, 131, 147 and 160. While Ser/Ala replacements of all six amino acids increased the association of REM6.6 with MTs and ATM1, Ser/Glu replacements decreased it dramatically. The double mutant plants of rem6.6/rem6.7 were similar to wild type. When the Rem6.6 cDNA, or the Ser/Ala or Ser/Glu constructs were expressed in rem6.6/6.7 plants under the 35S promoter, it was found that only the Ser/Glu Rem6.6 expressing plants exhibited a higher growth rate and a significantly bigger final size.