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. 2013 Feb 20;32(4):552-65.
doi: 10.1038/emboj.2012.354. Epub 2013 Jan 11.

Why Do Cellular Proteins Linked to K63-polyubiquitin Chains Not Associate With Proteasomes?

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Free PMC article

Why Do Cellular Proteins Linked to K63-polyubiquitin Chains Not Associate With Proteasomes?

James A Nathan et al. EMBO J. .
Free PMC article

Abstract

Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Proteins in lysate prevent proteasome binding to K63-polyUb chains. (A) Although pure proteasomes bind to K48 and K63 chains, proteasomes in the muscle lysate bind efficiently only to K48 conjugates. Ubiquitinated E6AP and Nedd4 were incubated with purified 26S particles and the bound proteasomes were measured by LLVY-AMC cleavage (right panel). These same ubiquitinated conjugates were incubated with a rat muscle extract (120 μg) (left panel), with or without the addition of Bortezomib (1 μM) and the proteasomes from the lysate that bound to the conjugates were measured by LLVY-AMC cleavage (Bortezomib residual activity was subtracted). (B) The cell lysate contains factors that reduce binding of K63, but not K48-polyUb conjugates to the proteasome. The proteasome depleted of rabbit muscle extract (200 μg) was incubated with the ubiquitinated substrates and proteasomes as shown in Supplementary Figure S1B. (C) DUB inhibitors do not influence the inhibition of K63-conjugate binding to proteasomes by cell extracts. Proteasome binding to the Ub conjugates was measured as in (B), with or without 4 mM N-ethylmaleimide (NEM) and 1 mM 1,10-o-phenanthroline (oPT). (D) Factors within the cell extract that inhibit the binding of K63 conjugates to the proteasomes, bind tightly to K63 chains and can be depleted from cell extracts. The rat muscle extract, ubiquitinated Nedd4 and 26S proteasomes were incubated as in (B). The unbound fraction of the lysate (flow-through) was then assayed for its capacity to block proteasome binding. The flow-through was incubated with a fresh column of ubiquitinated Nedd4 and proteasomes, and the conjugate bound proteasome fraction was then measured. (E) Multiple cellular proteins or complexes can prevent K63 conjugates binding to the proteasome. Proteins in the rat muscle lysate (4 mg/ml) were separated according to their molecular weight using a Sephacryl S300HR column. After a void volume of 7 ml, 0.5 ml lysate fractions were collected and incubated with the ubiquitinated Nedd4 and proteasomes, to assay their ability to block K63-conjugate binding. All values are the means±s.e.m. See also Supplementary Figure S2. E6, E6AP; N4, Nedd4.
Figure 2
Figure 2
Identification of K63- and K48-specific Ub-binding proteins. (A) Representative silver-stained gel of proteins eluted from K48 or K63 conjugates. After washing columns, proteins bound to the control or ubiquitinated substrates (E6AP and Nedd4) were eluted with His10-UIM, and 10% of eluted volume was separated by SDS–PAGE. (B) Identified UBD-containing proteins demonstrate specificity for K48 or K63 chains. Control or ubiquitinated substrates (E6AP and Nedd4) were incubated with an HEK293 lysate. Following washing, proteins were eluted from the resin in SDS-loading buffer and immunoblotted for the Rad23 proteins, TOM1, Hrs, and STAM. The size range of ubiquitinated E6AP and Nedd4 is shown with the anti-Ub antibody. *Non-specific band with anti-hHR23B antibody. (C) The ESCRT0 components, Hrs and STAM, bind specifically to K63 chains, while hHR23A and hHR23B are selective for K48 conjugates. Purified recombinant hHR23A, hHR23B, Hrs, and STAM at concentrations of 10, 50, and 100 nM were incubated with the ubiquitinated E6AP or Nedd4 resins. (D) HHR23A and B bind specifically to K48 chains. Resin bound GST-hHR23B (100 nM) was incubated with increasing concentrations (10, 50, and 100 nM) of K48- or K63-Ub tetramers in TBSG with 0.1% Triton X-100. Following washing, proteins were eluted from the resin and immunoblotted for hHR23B or Ub. (E) ESCRT0 preferentially binds to longer K63 chains. GST–Hrs and His–STAM were incubated with increasing concentrations of K63-Ub tetramers or K63 chains of mixed lengths (3–9mers), and immunoblotted for Ub (left panel). A Coomassie stained gel of the Ub-chain inputs is also shown (right panel). (F) K48 or K63 tetramers were forced onto GST–Nedd4 as described in Supplementary Figure S2. These conjugates were then incubated with hHR23A, hHR23B, and STAM (10, 50, and 100 nM) and the bound protein fraction visualized by immunoblotting.
Figure 3
Figure 3
ESCRT0 and its components selectively block K63-polyUb conjugate binding to the proteasome. (AD) Hrs, STAM, and the ESCRT0 complex inhibit K63 chains binding the 26S. Increasing concentrations of pure Hrs, STAM, TOM1, or ESCRT0 (Hrs and STAM used in equal amounts to allow formation of 1:1 complex) were incubated with the polyubiquitinated conjugates and 10 nM 26S particles. All values are the means±s.e.m. (E, F) Hrs and STAM (50 nM) were incubated with the 26S particles as described. Proteasome activity in these samples was measured by peptidase activity (E, left graph), and immunoblotting with densitometric quantification (ImageJ) of the 20S α-subunits (E, right graph and F).
Figure 4
Figure 4
HHR23A and B, by associating with K48 chains, increase proteasome binding to the K48 conjugates. (A) HHR23A and hHR23B stimulate K48-Ub conjugate binding to the 26S proteasome. The pure proteins were first incubated with ubiquitinated Nedd4 or E6AP for 30 min at 4°C. After washing, the resin was incubated with 10 nM 26S proteasomes for 30 min at 4°C, and their binding measured. (B) The combined presence of ESCRT0 and hHR23B together enhance the selectivity and also amount of binding of proteasomes to K48 chains. Polyubiquitinated Nedd4 or E6AP was incubated with 10 nM 26S proteasomes, and 50 nM ESCRT0, hHR23B or both together at 4°C for 30 min. All values are the means±s.e.m. See also Supplementary Figure S4.
Figure 5
Figure 5
Decreasing ESCRT0 levels in cells allows K63 conjugates to bind to the proteasome. (AC) HEK293T cells were transfected with or without siRNA targeting Hrs and STAM. After 72 h, the cells were lysed and depleted of endogenous 26S proteasomes in the usual manner. The lysates were then incubated with the pure ubiquitinated Nedd4 resins, and the amount of proteasomes bound to the resins measured. Schematic of the experimental method (A), representative immunoblots of STAM and Hrs knockdowns (B), and measurements of proteasomes bound to the K63 conjugates (C) are shown. β-actin served as a loading control (B, bottom panel). All values are the means±s.e.m.
Figure 6
Figure 6
ESCRT0 prevents polyubiquitinated MHC Class I molecules from binding to the proteasome in vivo. (A) The viral ligase K3 downregulates cell surface MHC Class I, which is partially rescued by knockdown of ESCRT0. HeLa and HeLa-K3 cells were transfected with siRNA targeted to STAM and Hrs. After 72 h, the levels of surface Class I were measured by flow cytometry. (B) ESCRT0 knockdown in HeLa-K3 cells partially rescues total MHC Class levels, but does not result in deubiquitination of Class I. HeLa and HeLa-K3 cells, transfected with siRNA targeting Hrs and STAM, were immunoprecipitated for Class I with a β2m antibody, and immunblotted for total Class I levels (HC10 antibody), STAM and Hrs. Polyubiqutinated Class I molecules were visualized on longer exposures (top panel), while shorter exposures show the partial rescue of Class I following Hrs and STAM knockdowns (middle and lower panels). The proteasome subunit, Rpt1, served as a loading control (bottom panel). (CE) Ubiquitinated cell surface MHC Class I associates with the 26S proteasome following a reduction in endogenous ESCRT0. HeLa and HeLa-K3 cells were transfected with siRNA against Hrs, STAM, or TSG101 in the usual manner. The cells were then labelled for 3 h with the conformational Class I antibody (W6/32) with or without 0.6 μM Bafilomycin A (Baf). These Class I molecules were immunoprecipitated, and the amount of proteasomes bound measured by LLVY-AMC peptidase activity. (C) Schematic diagram of the experiment. (D) Immunoblots of immunoprecipitated Class I (left), and lysate immunoblots of STAM, Hrs, TSG101, and β-actin (right). (E) Measurements of proteasomes bound. All values are the means±s.e.m.

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