The turnover of cell surface proteins of carrot protoplasts

The covalent modification of cell surface proteins with N-hydroxysuccinimid e eaters of biotin was used to develop a strategy for following the turnove r of proteins on the surface of carrot (Daucus carota L.) protoplasts. A bi otinylation/internalisation assay was established which enabled the turnove r of cell surface proteins to be examined by biochemical and immunocytochem ical techniques. The detection of biotinylated proteins after sodium dodecy l sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalent ly modified. Immunolocalisation of biotinylated proteins in protoplasts dir ectly after their derivatisation, demonstrated that the proteins were initi ally restricted to the cell surface. Incubation of biotinylated protoplasts at 25 degrees C for 1 h resulted in the detection of biotin-labelled prote ins on the cell surface and intracellularly. A small proportion of these: p roteins was associated with coated pits, the Golgi apparatus and vacuolar c ompartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate M-r 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of M-r 100 000 by an antibody ra ised against an isoform of a tobacco plasma-membrane H+-ATPase, strongly su ggests that the plasma-membrane H+-ATPase is internalised by carrot protopl asts. The implications of these results are discussed within the context of endocytosis in plants.