Endocytosis in proximal tubule cells involves a two-phase membrane-recycling pathway

The aims of the present study were to examine the initial events of endocytosis with respect to migration, internalization, and recycling of protein binding sites on the luminal plasma membrane of genuine renal proximal tubule cells (not cultured cells), and in particular the sequential involvement of dense apical tubules (DAT). Isolated rabbit proximal tubules were first perfused at low temperature (9 degrees C) with insulin-gold to label the binding sites and then with disuccinimidyl suberate (DSS) for 20 min to covalently cross-link insulin-gold and binding sites. The tubules were subsequently perfused for 4-30 min at 25 or 37 degrees C before fixation to follow the pathways of the binding sites. The present experimental approach allowed a temporal separation of the subcellular processes involved in endocytosis. The identity and organization of the different organelles were certified by serial sectioning of the experimental tubules. In tubules fixed directly at low temperature after cross-linking the label was located on the microvilli membranes and in plasma membrane invaginations. The labeling of microvilli decreased with time of perfusion at 25 degrees C in parallel with a simultaneous increase of labeling of invaginations, suggesting lateral migration of the binding sites. The invaginations pinched off from the surface to form endocytic vesicles. The binding sites were subsequently trafficked either to 1) tubular elongations from endocytic vesicles or small vacuoles, which again form DAT, or 2) in a later sequence trafficked to larger vacuoles, from which tubular elongations and DAT also form. The elongations formed the DAT that transported binding sites back to the luminal plasma membrane. Lysosomes and Golgi apparatus were not involved in membrane recycling. Thus the present study provides evidence for a two-phase recycling pathway of membrane proteins involved in endocytosis.