The turnover of cell surface proteins of carrot protoplasts

Abstract

The covalent modification of cell surface proteins with N-hydroxysuccinimide esters of biotin was used to develop a strategy for following the turnover of proteins on the surface of carrot (Daucus carota L.) protoplasts. A biotinylation/internalisation assay was established which enabled the turnover of cell surface proteins to be examined by biochemical and immunocytochemical techniques. The detection of biotinylated proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalently modified. Immunolocalisation of biotinylated proteins in protoplasts directly after their derivatisation, demonstrated that the proteins were initially restricted to the cell surface. Incubation of biotinylated protoplasts at 25 °C for 1 h resulted in the detection of biotin-labelled proteins on the cell surface and intracellularly. A small proportion of these proteins was associated with coated pits, the Golgi apparatus and vacuolar compartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate Mr 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of Mr 100 000 by an antibody raised against an isoform of a tobacco plasma-membrane H+-ATPase, strongly suggests that the plasma-membrane H+-ATPase is internalised by carrot protoplasts. The implications of these results are discussed within the context of endocytosis in plants.