STUDIES OF TRANSFERRIN RECYCLING RECONSTITUTED IN STREPTOLYSIN-O PERMEABILIZED CHINESE-HAMSTER OVARY CELLS

Efficient transferrin receptor recycling is reconstituted when donor c ytosol and ATP are added to the streptolysin O permeabilized cells. Th e rate of reconstituted recycling is dependent on the concentration of donor cytosol. The cytosol provides a factor(s) required for the tran sport of transferrin from the pericentriolar recycling compartment to the plasma membrane. N-Ethylmaleimide treatment of permeabilized cells inhibits both the fusion of recycling vesicles with the plasma membra ne as well as the formation of functional recycling vesicles from the pericentriolar recycling compartment. Guanosine 5'-3-O-(thio)triphosph ate (GTP gamma S) does not affect reconstituted recycling in the prese nce of an optimal cytosol concentration. Therefore, the rate-limiting step in recycling is not regulated by GTP-hydrolyzing proteins, and hy drolysis of GTP is not required for endocytic recycling. GTP gamma S s timulates recycling when suboptimal concentrations of cytosol are used . This stimulatory effect is not mediated by a brefeldin A-sensitive A DP-ribosylation factor protein. Addition of mild-type donor cytosol to permeabilized END2 Chinese hamster ovary cells, which recycle transfe rrin at half the rate of mild-type cells, reconstitutes recycling to t he reduced rate of intact END2 cells but not to the mild-type recyclin g rate. These results indicate that the defect responsible for the slo wed transferrin recycling in END2 mutants is membrane associated or th at the defective protein is too large to diffuse out of the cells thro ugh the streptolysin O pores.