Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants

Abstract

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gβ). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 °C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.