Domain studies on Remorin AtREM1.3 intrinsic disorder

Remorins are plant-specific proteins, that may act as scaffold proteins for signalling complexes during plant innate immunity and microbial invasion of host cells [1, 2]. Several members of this protein family associate with membrane rafts [3,4], can bind DNA [5] and form filaments in vitro [6]. All remorins exhibit a conserved C-terminal region harbouring a predicted coiled-coil motif and an evolutionary divergent N-terminal region [7]. Additionally, a novel plasma membrane (PM) anchoring motif has been identified within the C-terminal region [unpublished data]. To get insights into the function of the different regions, we cloned, expressed and purified different truncation, alternative splicing and phosphomimetic variants of the Remorin AtREM1.3 and characterized their cellular localization and structural properties. Using circular dichroism (CD), limited proteolysis (MS mapping of resistant fragments) and size exclusion chromatography we found that AtREM1.3 shows a high degree of intrinsic disorder, which is concentrated in the N-terminal region. Additionally, as in other intrinsically disordered proteins, increasing concentrations of TFE cause an increase in α helical structure. The ability of the different variants to bind DNA and other polyanions as well as their ability to be phosphorylated by different kinases involved in plant immunity has been studied in vitro. Finally we have localized the different variants in planta showing that removal of the putative PM anchoring motif and the N-terminal region confers nuclear and nucleolar localization.

The high degree of intrinsic disorder in the N-terminal region further supports the idea that remorins may act as scaffold proteins at the PM. However, DNA-binding activity and the intrinsic affinity of truncated variants for nuclear localization open several questions regarding a possible role in this compartment.