A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting

Abstract

In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.

FIGURE 1:

K63Ub chains are required for efficient sorting to yeast MVBs. SUB cells genetically modified to express only plasmid-encoded wt or single-KR-mutant Ub were transformed with a plasmid encoding Ear1-mCherry (mCh) under the control of the GPD1 promoter. (A) Ear1-mCherry ubiquitylation profile. Total protein extracts separated by SDS–PAGE were immunoblotted with an anti-DsRed antibody. The arrow and arrowhead indicate the monoubiquitylated and polyubiquitylated forms of Ear1-mCherry, respectively. The anti-Pgk1 blot serves as a loading control. (B) Ear1-mCherry sorting to the MVB pathway was assessed by fluorescence microscopy.

FIGURE 2:

K63-linked chain-modified proteins accumulate in the class E compartment. (A) Yeast cells expressing plasmid-encoded wt or K63R Ub as sole source of Ub were analyzed by IF microscopy using an anti-K63Ub antibody. No specific labeling of any cellular structure was observed. (B) Localization of the MVB cargo Sit1-GFP in vps23Δ cells by fluorescence microscopy. The arrow and arrowhead show the presence of Sit1-GFP at the plasma and vacuolar membrane, respectively. (C) vps23Δ cells expressing plasmid-encoded wt Ub or UbK63R as the sole source of Ub and producing GFP-Phm5 were analyzed by IF microscopy with anti-K63Ub and anti-GFP antibodies. (D) vps23Δ cells expressing plasmid-encoded wt Ub or UbK63R as the sole source of Ub were analyzed by IF microscopy with anti-K63Ub or anti-K48Ub antibodies, combined with DAPI staining. The anti-K63Ub antibody labeled specifically and homogeneously the class E compartment in all cells. The vacuolar membrane (arrowhead) or the plasma membrane (arrow) were sporadically stained. (E) Serial dilutions of liquid cultures of cells expressing wt Ub (SUB280 and vps23Δ) or UbK63R (SUB413) transformed with an empty URA3 plasmid (pRS316) were grown in solid YNB medium supplemented with 2% glucose in the presence or absence of toxic plasma membrane transporter substrates at the following concentrations: 5 μg/ml 5-FU, 10 μg/ml ethionine, 0.5 μg/ml canavanine, and 5 μM cadmium.

FIGURE 3:

The human melanosomal protein MART-1 is modified by K63Ub chains. (A) Lysates from human melanocytic MNT-1 cells were immunoprecipitated with an anti-IgG2B (control) or anti-MART-1 antibody and immunoblotted with anti-MART-1, anti-Ub, anti-K63Ub, or anti-K48Ub antibodies. Arrows indicate ubiquitylated forms of MART-1 modified by K63Ub chains. (B–G) HeLa cells transfected with MART-1 were analyzed by IF microscopy with antibodies against MART-1 (C, F) and K63Ub chains (B, E). Overlays are shown in D and G. E–G correspond to a 1.8× magnification of the boxed areas in B–D, respectively.

FIGURE 4:

The inhibition of K63Ub chain formation impairs MART-1 endosomal sorting. (A, B) HeLa cells were transfected with plasmids encoding MART-1 and GFP-tagged wt Ub (Ub^wt-GFP) or UbK63R (Ub^K63R-GFP). Ultrathin cryosections were double immunogold labeled with anti–MART-1 (15-nm gold particles, arrow) and anti-GFP (10-nm gold particles, arrowhead) antibodies. (C) Quantification of the various sorting phenotypes of MART-1 as a percentage of gold particles per membrane compartment. EE, early endosomes; ILVs, intraluminal vesicles of the MVBs; LM, limiting membrane of the MVBs. Asterisk indicates deviation <0.05.

FIGURE 5:

Ub fusion to the cargo only partially bypasses the MVB sorting defect in cells unable to synthesize K63Ub chains. (A) GFP-Phm5 was expressed in yeast expressing plasmid-encoded wt Ub (SUB280) or UbK63R as the sole source of Ub (SUB413). The efficiency of sorting to the vacuolar lumen was assessed by fluorescence microscopy. (B) Ub-GFP-Phm5-K6R was expressed in the same cells and observed by fluorescence microscopy. (C) Quantification of the various sorting phenotypes observed in B. The numbers indicate the percentage of the total cell population of 700 U. VL, vacuolar lumen; VM, vacuolar membrane. The heterogeneity of the observed sorting phenotypes is probably due to differences in the levels of Ub and cargo in individual cells.

FIGURE 6:

All UBD-containing ESCRT proteins are ubiquitylated. (A) Schematic representation of UBD-containing ESCRT proteins drawn to scale. (B) Cell lysates from wt, rsp5, or ubp2Δ strains (BY4741 genetic background) expressing Hse1-3xHA, 6xHA-Vps27, Vps36-3xHA, or Bro1-3xHA and overproducing 6His-Ub were passed through nickel columns. Lysates (L) and eluates (E) were subjected to SDS–PAGE and immunoblotted with an anti-HA antibody. Bars and arrows indicate the ubiquitylated forms of each protein. Asterisks indicate ubiquitylated forms of Vps36-3xHA specifically enriched in ubp2Δ cells. (C) The same experiment as in B was performed for ubp2Δ cells expressing Vps23-3xHA. (D) Serial dilutions of liquid cultures of 23344c (wt), 27038b (npi1/rsp5), and VA029 (ubp2Δ) cells were grown on solid YNB medium plus 2% glucose supplemented with uracil in the presence or absence of toxic plasma membrane transporter substrates, as described in Figure 2E.

FIGURE 7:

An inability to assemble or disassemble K63Ub chains affects MVBs biogenesis and ultrastructure. Ultrastructure of endosomal compartments in vam3-ts cells expressing wt or K63R Ub at the restrictive temperature. Nanogold particle uptake was assessed with spheroplasts obtained from RHT514, RHT515, and RHT516 cells before EM analysis. All the compartments accessible to the Nanogold particles were defined and subdivided into classes on the basis of morphological criteria (see Materials and Methods). (A) MVBs observed in RHT514 cells. (B) MVBs detected in preparations from the RH514 strain in which no silver enhancement reaction was performed, providing a clearer illustration of the morphological details of this organelle. (C) An example of a type1 EE from RHT514 cells. (D) The ultrastructure of a type 2 EE on sections from the RH514 strain. (E) A remnant MVB observed in the RHT515 mutant. (F, F′) Remnant MVBs from a preparation in which no silver enhancement reaction was performed. (G) An example of a type1 EE detected in RHT515 cells. (H) A type 2 EE in the RH515 mutant. (I) MVBs observed in the ubp2∆ vam3ts mutant. (J) Statistical evaluation of the EM preparations. MVBs, type 1 and type 2 EEs, and remnant MVBs (rMVBs) were counted in 100 randomly chosen cell profiles in two independent experiments. Error bars represent the standard deviations, which were also used for a t test, confirming the statistical significance of the data (p < 0.05). Bar, 100 nm.