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Vol. 13, Issue 1, 169-182, January 2002
Department of Cell Biology, University Medical Center and Institute
of Biomembranes, Utrecht University, 3584 CX Utrecht, The Netherlands
Previously we described clathrin-coated buds on tubular early
endosomes that are distinct from those at the plasma membrane and the
trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a
temperature-sensitive mutant of dynamin-1 (dynamin-1G273D)
or, as a control, dynamin-1 wild type. In
dynamin-1G273D-expressing cells, 29-36% of endocytosed
transferrin failed to recycle at the nonpermissive temperature and
remained associated with tubular recycling endosomes. Sorting of
endocytosed transferrin from fluid-phase endocytosed markers in early
endosome antigen 1-labeled sorting endosomes was not inhibited.
Dynamin-1G273D associated with accumulated clathrin-coated
buds on extended tubular recycling endosomes. Brefeldin A interfered
with the assembly of clathrin coats on endosomes and reduced the extent
of transferrin recycling in control cells but did not further affect
recycling by dynamin-1G273D-expressing cells. Together,
these data indicate that the pathway from recycling endosomes to the
plasma membrane is mediated, at least in part, by endosome-derived
clathrin-coated vesicles in a dynamin-dependent manner.
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