AbstractsBiology & Animal Science

Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated vesicles fuse, after uncoating, with sorting endo-somes. In this organelle recycling proteins are sorted from lysosomally directed proteins. Recycling is thought to occur either directly from sorting endosomes to the plasma membrane or via a second compartment that is formed in the perinuclear area and is composed of tubular recycling endosomes. The nature of the putative recycling vesicles that derive from these two endosome populations was unknown, but it has been suggested that endocytosed membrane constituents recycle by default. However, in a previous study we characterized a novel class of clathrin coated buds on recycling endosomes (Stoorvogel et al., 1996. J. Cell Biol. 132, 21-33), and proposed that clathrin-coated vesicles might be involved in the recycling pathway from sorting endosomes. In chapter 2 we now show that these endosome associated clathrin-coated buds contain dynamin-2, a GTPase that already had an established function in the fission of clathrin-coated vesicles at the plasma membrane. To study recycling processes, we monitored the trafficking of the transferrin receptor (TfR), a prototype recycling protein, in cells which overexpress a tem-perature- sensitive dynamin-1 mutant (dyn ts ). At the non-permissive temperature, ~30% of endocytosed transferrin (Tf) was retained in recycling endosomes by dyn ts cells. At these same conditions dynamin-labeled clathrin-coated buds accumulated on tubular TfR-containing endo-somes. In addition, recycling endosomes formed a more elaborate tubular network, suggesting that fission of TfR containing clathrin-coated vesicles from tubular endosomes requires func-tional dynamin. In contrast, exit from sorting endosomes was normal in these cells. From these results, it was concluded that TfR recycling from recycling endosomes to the plasma membrane is mediated by clathrin-coated vesicles and requires a functional dynamin, while exit from sort-ing endosomes is a dynamin-independent process. In chapter 3 we made use of the reversible phosphatidyl inositide (PI) 3-kinase inhibitor LY294002 to study the requirements for PI 3-kinase in TfR recycling. LY294002 did not inter-fere with the entry of TfR into sorting endosomes, nor with transport to, or release from recy-cling endosomes. However, egress of endocytosed Tf from sorting endosomes was significant-ly delayed. LY294002 and dyn ts had synergistic effects on Tf recycling kinetics, indicating that 98?they interfered with two distinct recycling pathways which can partly compensate for each oth-ers loss of function. The inhibitory effect of LY294002 on Tf recycling was reversed upon removal of the drug, indicating that its inhibitory effect was not due to delayed transport from sorting endosomes to recycling endosomes. In…