SORTING OF MEMBRANE-COMPONENTS FROM ENDOSOMES AND SUBSEQUENT RECYCLING TO THE CELL-SURFACE OCCURS BY A BULK FLOW PROCESS

A central question in the endocytic process concerns the mechanism for sorting of recycling components (such as transferrin or low density l ipoprotein receptors) from lysosomally directed components; membrane-a ssociated molecules including receptors are generally directed towards the recycling pathway while the luminal content of sorting endosomes, consisting of the acid-released ligands, are lysosomally targeted. Ho wever, it is not known whether recycling membrane receptors follow bul k membrane flow or if these proteins are actively sorted from lysosoma lly directed material because of specific protein sequence and/or stru ctural features. Using quantitative fluorescence microscopy we have de termined the endocytic route and kinetics of traffic of the bulk carri er, membrane lipids, to address this issue directly. We show that epsi lon-aminohexanoyl]-sphingosylphosphorylcholine (C6-NBD-SM) in endocyto sed as bulk membrane, and it transits the endocytic system kinetically and morphologically identically to fluorescently labeled transferrin in a CHO cell line. With indistinguishable kinetics, the two labeled m arkers sort from lysosomally destined molecules in peripherally locate d sorting endosomes, accumulate in a peri-centriolar recycling compart ment, and finally exit the cell. Other fluorescently labeled lipids, C 6-NBD-phosphatidylcholine and galactosylceramide also traverse the sam e pathway. The constitutive nature of sorting of bulk membrane towards the recycling pathway and the lysosomal direction of fluid phase impl ies a geometric basis of sorting.