Isolation of Plasma Membrane and Plasma Membrane Microdomains.

The plasma membrane surrounds the cytoplasm of a cell and functions as a barrier to separate the intracellular compartment from the extracellular environment. Protein and lipid components distribute nonuniformly and the components form clusters with various functions in the plasma membrane. These clusters are called as "microdomains." In plant cells, microdomains have been studied extensively because they play important roles in biotic/abiotic stress responses, cellular trafficking, and cell wall metabolism. Here we describe a standard protocol for the isolation of the plasma membrane and microdomains from plant cells, Arabidopsis and oat.

Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.

Detergent-resistant plasma membrane proteome to elucidate microdomain functions in plant cells.

Although proteins and lipids have been assumed to be distributed homogeneously in the plasma membrane (PM), recent studies suggest that the PM is in fact non-uniform structure that includes a number of lateral domains enriched in specific components (i.e., sterols, sphingolipids, and some kind of proteins). These domains are called as microdomains and considered to be the platform of biochemical reaction center for various physiological processes. Microdomain is able to be extracted as detergent-resistant membrane (DRM) fractions, and DRM fractions isolated from some plant species have been used for proteome and other biochemical characterizations to understand microdomain functions. Profiling of sterol-dependent proteins using a putative microdomain-disrupting agent suggests specific lipid-protein interactions in the microdomain. Furthermore, DRM proteomes dynamically respond to biotic and abiotic stresses in some plant species. Taken together, these results suggest that DRM proteomic studies provide us important information to understand physiological functions of microdomains that are critical to prosecute plant's life cycle successfully in the aspect of development and stress responses.