Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation

Abstract

The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.

Figure 1

Hse1 fused to a deubiquitinating enzyme blocks MVB sorting of ubiquitinated proteins. (A) Schematic of the Hse1-DUb fusion protein showing key functional domains of the Hse1 subunit of ESCRT-0 fused to the catalytic domain of the deubiquitinating enzyme UL36. VHS (Vps27, Hrs, STAM), UIM (Ub-interacting motif) and SH3 (Src-homology 3). (B) pep4Δ cells expressing either empty vector (−) or GFP-Ub (+) were grown to log phase and radiolabelled with three 5-min pulses of ³⁵S-methionine before spheroplasting and lysis; lysates were then subjected to immunoprecipitation using anti-GFP antibodies. (C) Localization of GFP-Ub in WT cells or WT cells carrying integrated CUP1-Hse1-DUb (upper panels). Cells were grown with CuCl₂ for 7 h before microscopy. Lower panels show localization of GFP-Ub in vps4Δ with or without expression of Hse1-DUb. Cells were counter-labelled with the lipid-binding endocytic tracer FM4-64 (at 30 °C for 30 min), and then chased in rich media for 15 min. Accumulation of FM4-64 within enlarged endosomal ‘class E compartments’ (arrowheads) was clearly observed in vps4Δ mutants. Scale bar, 5 μm. (D) Class E compartment endosomes (identified by accumulation of FM4-64) from 275 mutant vps4Δ cells or 101 vps4Δ cells expressing Hse1-DUb were scored for colocalization with GFP-Ub. Bars represent the mean and error bars s.d. ESCRT, Endosomal Sorting Complexes Required for Transport; GFP, green fluorescent protein; MVB, multivesicular body; Ub, ubiquitin; WT, wild type.

Figure 2

Loss of ubiquitinated cargo abolishes the generation of intralumenal vesicles. (A) Schematic illustration showing forms of cargo (Mup1-GFP) with or without fusion to Ub, and forms of the ESCRT-0 subunit Hse1 with or without fusion to the UL36 DUb enzyme, which were used in each experimental group. (B) Localization of Mup1-GFP in pep4Δ atg8Δ cells without or with the expression of Hse1-DUb. Localization of Mup1-GFP-Ub in pep4Δ atg8Δ Hse1-DUb cells is also shown (right panels). Cells were grown in rich media (YPD replete with methionine) for 12 h in the presence of CuCl₂. Scale bar, 5 μm. (C) TEM-based analysis of the cells shown in B. Cells were spheroplasted, fixed, stained and analysed by TEM. V, M and N are labelled. Scale bar, 1 μm. (D) Cryo-electron microscope-based analysis of cells in B. Scale bar, 1 μm. (E) Magnification of cryo-electron microscope images of MVB-localized ILVs in D. Scale bar, 50 nm. DUb, deubiquitinating; ESCRT, Endosomal Sorting Complexes Required for Transport; GFP, green fluorescent protein; ILV, intralumenal vesicle; M, mitochondria; MVB, multivesicular body; N, nuclei; TEM, transmission electron microscope; Ub, ubiquitin; V, vacuoles.

Figure 3

Additional ubiquitinated cargo replenishes accumulation of ILVs. (A) Morphometric analysis of TEM experiment described in Fig 2C. Average number of ILVs (per cell per section) as calculated by counting ILVs within the vacuoles of 24 wild-type cells, 52 wild-type cells expressing Hse1-DUb and 36 wild-type cells coexpressing Hse1-DUb and Mup1-GFP-Ub. Error bars=s.d. Average vesicle size was calculated from measurements of 329 ILVs from wild-type cells, and 333 ILVs from cells coexpressing Hse1-DUb and Mup1-GFP-Ub. Error bars=s.d. (B) TEM images of vma4Δ cells with and without expression of Hse1-DUb. Cells were fixed before spheroplasting, imbedding and electron microscopy analysis. (C) Fluorescence localization and TEM analysis of WT cells expressing Mup1-GFP-Ub alone or expressing Mup1-GFP and Hse1-dub^* in which the DUb carries an inactivating mutation, and cells expressing Hse1-DUb (active) that coexpress either Ste3-GFP-Ub or Ub-GFP-Cps1. DUb, deubiquitinating; ESCRT, Endosomal Sorting Complexes Required for Transport; GFP, green fluorescent protein; ILV, intralumenal vesicle; TEM, transmission electron microscopy; Ub, ubiquitin; V, vacuoles.

Figure 4

Effect of Ub-cargo on ESCRT localization to endosomes. (A) Localization of GFP-tagged subunits of ESCRT-0 (Vps27), ESCRT-I (Vps23 and Vps28) and ESCRT-II (Vps22 and Vps36) in wild-type cells in the absence or presence of Hse1-DUb. Cells were grown in minimal media in the presence of CuCl₂ for 7 h before microscopy. Localization of GFP-tagged ESCRTs in Hse1-Dub-expressing cells that were coexpressing Mup1-RFP-Ub is also depicted (right hand columns). Scale bar, 5 μm. (B) Localization of GFP-tagged ESCRT subunits in vps4Δ cells in the absence or presence of Hse1-DUb expression. Scale bar, 5 μm. DUb, deubiquitinating; ESCRT, Endosomal Sorting Complexes Required for Transport; GFP, green fluorescent protein; Ub, ubiquitin; WT, wild type.

Figure 5

Function and Functional modelling of GFP-tagged ESCRTs. (A) WT cells or cells expressing the indicated GFP-tagged ESCRT subunit as their sole source of that protein were analysed for proper MVB sorting of Sna3-RFP to the vacuole interior. Also shown is the same analysis of Vps28-GFP and Vps36-GFP cells lacking VPS4. (B) CPY was immunoprecipitated from intracellular and secreted extracellular fractions from cells pulse-labelled with ³⁵S-methionine for 10 min and chased for 50 min at 30 °C. (C) Model for how Ub-cargo could drive ESCRT recruitment or retention and subsequent ILV formation. In the absence of cargo, ESCRTs rapidly recycle off endosomes and are unable to form a productive complex. In the presence of cargo, ESCRTs are retained better, allowing them to organize properly and complete the formation of ILVs while concomitantly sorting Ub-cargo into those vesicles. CPY, carboxypeptidase Y; E, extracellular; ESCRT, Endosomal Sorting Complexes Required for Transport; GFP, green fluorescent protein; I, intracellular; ILV, intralumenal vesicle; MVB, multivesicular body; RFP, red fluorescent protein; Ub, ubiquitin; WT, wild type.