Molecular mechanism of multivesicular body biogenesis by ESCRT complexes - PubMed (original) (raw)

Molecular mechanism of multivesicular body biogenesis by ESCRT complexes

Thomas Wollert et al. Nature. 2010.

Abstract

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the endosomal sorting complex required for transport (ESCRT) complexes 0, I, II and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inwards by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, -I, -II and -III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. Here we show that ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and ESCRT-II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and ESCRT-II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.

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Figures

Fig. 1

Fig. 1

Model cargo clustering by ESCRT-0. a, ESCRT-0 induces the formation of Ub domains. (b) Ub mutant I44D is not clustered under the same conditions as in (a). Tagged Ub fusions were present at 65 nM. (c) Clusters of ESCRT-0 (15 nM, labeled with Alexa 488) can form in the presence of Ub I44D, but the size and number of clusters is reduced. The presence of 15 nM ESCRT-I and II has little effect on the clustering. (d) Ub-CFP clustering by ESCRT-0 is prevented in the I44D mutant. Error bars in (c) and (d) were calculated from the standard deviation of n = 3 separate experiments. Scale bar = 10 µm.

Fig. 2

Fig. 2

ESCRT-I and –II-induce membrane buds and confine cargo in them. Labeled ESCRT-I and unlabeled ESCRT-II (a) and vice versa (b) induce buds, as summarized (c). (d, e) Fluorescence recovery after photobleaching (FRAP) experiments of model cargo confined in buds. Arrows indicate buds in which Ub-GFP does not significantly recover on the time scale of the experiment. Error bars in were calculated from the standard deviation of n = 3 separate experiments in part (c) and for n = 10 different buds each selected from a different GUV in (e). Scale bar = 10 µm.

Fig. 3

Fig. 3

ESCRT-I and II localize to the necks of membrane buds. Imaging was carried out in the presence of 15 nM unlabeled Vps20, which binds to ESCRT-II and augments ESCRT-I and II occupancy at the neck. ESCRT-I and –II were present at 15 nM each in both experiments. Labeled ESCRT-I in the presence of unlabeled ESCRT-II (a) and labeled ESCRT-II in the presence of unlabeled ESCRT-I (b) induce membrane buds and co-localize with their necks. Scale bar = 10 µm and 2 µm (insets).

Fig. 4

Fig. 4

ESCRT-0 Ub domains co-localize with ESCRT-I-II membrane buds. (a) Wildtype yeast ESCRT-0 (15 nM, Alexa 488-labelled) in the presence of 65 nM His6-Ub-CFP colocalizes with buds induced by 15 nM each unlabelled ESCRT-I and –II. The second row shows a close-up of an individual cluster-bud pair. (b) ESCRT-0 mutated in three P(S/T)XP motifs required for ESCRT-I binding does not colocalizes with membrane buds. (c) Results for 70 GUVs are summarized in histograms. Error bars in were calculated from the standard deviation of n = 3 separate experiments. Scale bar = 10 µm (upper panels showing entire GUV) and 2 µm (inset).

Fig. 5

Fig. 5

ESCRT-III localizes to bud necks for membrane scission. 15 nM each of unlabeled ESCRT-0, ESCRT-I, and ESCRT-II, and 65 nM of His-Ub-GFP were present in both experiments. a,Vps20 (15 nM, labeled with Alexa 488) is localized to bud necks but does not sever the necks. b, Snf7 (15 nM, labeled with Alexa 488) localizes to bud necks when Vps20 (15 nM, unlabeled) is present. (c) The dependence of ILV production on Snf7 is indicated by the histogram of observations on 100 GUVs. Error bars in were calculated from the standard deviation of n = 3 separate experiments. Scale bar = 10 µm (upper panels showing entire GUV) and 2 µm (inset).

Fig. 6

Fig. 6

Molecular mechanism of MVB biogenesis. a, ESCRT-0 self-assembles and clusters cargo. b, The ESCRT-I and –II contain multiple membrane binding sites separated by a rigid stalk in ESCRT-I and a rigid Y-structure in ESCRT-II that could prop open membrane necks. c, ESCRT-II recruits Vps20 to the neck, which in turn recruits Snf7, the main engine for neck scission. d, Following scission, cargo is internalized in ILVs while ESCRT-III remains on the outside of the limiting membrane until it is recycled by Vps4.

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