We have used an in vitro fusion assay to study the mechanisms of transport from early to late endosomes. Our data show that the late endosomes share with the early endosomes a high capacity to undergo homotypic fusion in vitro. However, direct fusion of early with late endosomes does not occur. We have purified vesicles which are intermediates during transport from early to late endosomes in vivo, and analyzed their protein composition in two-dimensional gels. In contrast to either early or late endosomes, these vesicles do not appear to contain unique proteins. Moreover, these vesicles undergo fusion with late endosomes in vitro, but not with each other or back with early endosomes. In vitro, fusion of these endosomal vesicles with late endosomes is stimulated by polymerized microtubules, consistent with the known role of microtubules during early to late endosome transport in vivo. In contrast, homotypic fusion of early or late endosomes is microtubule-independent. Finally, this stimulation by microtubules depends on microtubule-associated proteins and requires the presence of the minus-end directed motor cytoplasmic dynein, but not the plus-end directed motor kinesin, in agreement with the microtubule organization in vivo. Our data strongly suggest that early and late endosomes are separate, highly dynamic organelles, which are connected by a microtubule-dependent vesicular transport step.

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

In the present paper, we show that transport from early to late endosomes is inhibited at the restrictive temperature in a mutant CHO cell line (ldlF) with a ts-defect in ε coatomer protein (εCOP), although internalization and recycling continue. Early endosomes then appear like clusters of thin tubules devoid of the typical multivesicular regions, which are normally destined to become vesicular intermediates during transport to late endosomes. We also find that the in vitro formation of these vesicles from BHK donor endosomes is inhibited in cytosol prepared from ldlF cells incubated at the restrictive temperature. Although εCOP is rapidly degraded in ldlF cells at the restrictive temperature, cellular amounts of the other COP-I subunits are not affected. Despite the absence of εCOP, we find that a subcomplex of β, β′, and ζCOP is still recruited onto BHK endosomes in vitro, and this binding exhibits the characteristic properties of endosomal COPs with respect to stimulation by GTPγS and sensitivity to the endosomal pH. Previous studies showed that γ and δCOP are not found on endosomes. However, αCOP, which is normally present on endosomes, is no longer recruited when εCOP is missing. In contrast, all COP subunits, except obviously εCOP itself, still bind BHK biosynthetic membranes in a pH-independent manner in vitro. Our observations thus indicate that the biogenesis of multivesicular endosomes is coupled to early endosome organization and depends on COP-I proteins. Our data also show that membrane association and function of endosomal COPs can be dissected: whereas β, β′, and ζCOP retain the capacity to bind endosomal membranes, COP function in transport appears to depend on the presence of α and/or εCOP.

Endomembrane trafficking is essential for all eukaryotic cells. The best-characterized membrane trafficking organelles include the endoplasmic reticulum (ER), Golgi apparatus, early and recycling endosomes, multivesicular body, or late endosome, lysosome/vacuole, and plasma membrane. Although historically plants have given rise to cell biology, our understanding of membrane trafficking has mainly been shaped by the much more studied mammalian and yeast models. Whereas organelles and major protein families that regulate endomembrane trafficking are largely conserved across all eukaryotes, exciting variations are emerging from advances in plant cell biology research. In this review, we summarize the current state of knowledge on plant endomembrane trafficking, with a focus on four distinct trafficking pathways: ER-to-Golgi transport, endocytosis, trans-Golgi network-to-vacuole transport, and autophagy. We acknowledge the conservation and commonalities in the trafficking machinery across species, with emphasis on diversity and plant-specific features. Understanding the function of organelles and the trafficking machinery currently nonexistent in well-known model organisms will provide great opportunities to acquire new insights into the fundamental cellular process of membrane trafficking.