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. 2006 Dec 4;175(5):715-20.
doi: 10.1083/jcb.200606113. Epub 2006 Nov 27.

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

Daniel P Nickerson  1 , Matthew West, Greg Odorizzi
Affiliations

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

Daniel P Nickerson et al. J Cell Biol. .

Erratum in

  • J Cell Biol. 2006 Dec 18;175(6):1043

Abstract

The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.

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Figures

Figure 1.
Figure 1.
The C terminus of Did2 binds Vps4. (A) Domain maps of Did2 and Vps4. (B and C) Western blots of in vitro glutathione-Sepharose pull downs of wild-type or mutant His6-Vps4 mixed with GST-Did2 (B) or wild-type His6-Vps4 mixed with wild-type or mutant GST-Did2 (C). (D) Fluorescence and DIC microscopy of FM 4-64–stained cells expressing GFP-Vps4E233Q. Arrowheads indicate endosomal membranes.
Figure 2.
Figure 2.
The N terminus of Did2 requires the Vps2–Vps24 subcomplex for recruitment to endosomes. (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells expressing GFP-tagged wild-type (A) or mutant (B) Did2 proteins. Arrowheads indicate endosomal membranes. (C and D) Subcellular fractionation and Western blot analysis of cells expressing GFP-tagged wild-type (C) or mutant (D) Did2 proteins. Cell lysates (T, total) were separated into membrane-associated pellet (P13) and soluble cytosolic (S13) fractions. PGK, 3-phosphoglycerate kinase. (E) In vitro glutathione-Sepharose pull downs of His6-Vps24 with GST-tagged wild-type or mutant Did2 proteins.
Figure 3.
Figure 3.
Did2 is required for the endosomal dissociation of ESCRT-III. (A and B) Fluorescence and DIC microscopy of FM 4-64–stained cells expressing Vps23- (A) or Vps36-GFP (B). Arrowheads indicate class E compartments. (C) Subcellular fractionation and Western blot analysis of endogenous Snf7 and Vps24. T, total; PGK, 3-phosphoglycerate kinase.
Figure 4.
Figure 4.
Tomographic analysis of endosome morphology. (A–I) Two-dimensional cross sections and three-dimensional models derived from 200-nm–thick section tomograms. V, vacuole. Models depict a wild-type MVB (B and C), a vps4Δ class E compartment (E and F), and a did2Δ VTE (H and I). Models in C, F, and I are rotated 25° relative to the models in B, E, and H. In MVB and VTE models, the endosomal limiting membrane is yellow, and lumenal vesicles are red. Cisternae in the vps4Δ class E compartment model are depicted in various colors to easily discriminate individual membrane structures. (J) Vesicle diameters in wild-type and did2Δ cells (n = 284 and 175, respectively). Mean values ± SEM (error bars; unpaired t test; P < 0.0001). (K) Fluorescence and DIC microscopy of cells expressing GFP-CPS.
Figure 5.
Figure 5.
Model of Did2 function in MVB sorting. The Did2 C terminus interacts with Vps4, and its N terminus interacts with the ESCRT-III subunit Vps24. Did2 is required for the endosomal dissociation of ESCRT-III and downstream components, which are Did2-dependent Vps4 substrates (green). ESCRT-I and -II (orange) are Did2-independent Vps4 substrates.
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