Protein targeting and degradation are coupled for elimination of mislocalized proteins

Abstract

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.

Fig. 1. Non-translocated PrP is rapidly ubiquitinated

(a) PrP translations in reticulocyte lysate, without or with rough microsomes (RMs), were analyzed directly (left) or after isolation of ubiquitinated products (right). (b) Time course of PrP synthesis and ubiquitination in vitro. (c) PrP containing (‘term.’) or lacking (‘trunc.’) a termination codon was translated in vitro. Truncated PrP was released with puromycin without or with cytosol and ubiquitination analyzed. Arrowhead indicates tRNA-containing PrP, which can be digested by RNAse. (d) Wild type PrP or constructs lacking the signal sequence (ΔSS) or both the signal sequence and GPI anchor (ΔSSΔGPI) were analyzed for ubiquitination. Prl-SS and NYP-SS contain signals from Prolactin and Neuropeptide Y, respectively.

Fig. 2. Bag6 interacts with MLPs via hydrophobic domains

(a) PrP translated in RRL or Fr-RRL, without or with 10 uM ubiquitin, was analyzed directly (left) or after anti-ubiquitin immunoprecipitation (right). (b) PrP translated in Fr-RRL is ubiquitinated when UbcH5a (E2; 250 nM) is included co-translationally or added post-translationally. Total synthesis (bottom) and ubiquitinated products (top) are shown. (c) PrP was immunoaffinity purified under native conditions, and incubated with the indicated components (‘cyt’ is cytosol; E1 was at 100 nM; E2 was UbcH5a at 250 nM). All reactions contained His-ubiquitin and ATP. Purified ubiquitinated products are shown. (d) PrP translated in Fr-RRL was separated into 10 fractions on a 5–25% sucrose gradient and subjected to chemical crosslinking (bottom gel) or ubiquitination assays (top graph). Asterisks indicate crosslinks. (e) Crosslinking reactions of in vitro synthesized PrP or deletion constructs were analyzed directly or after immunoprecipitation.

Fig. 3. Bag6 captures MLPs released from the ribosome

(a) Diagram of constructs derived from Sec61β, with transmembrane domains shown as grey boxes and hydrophilic changes in white boxes. (b) RNCs of β-CFP with the TMD outside the ribosome were subjected to crosslinking before or after release with puromycin, and analyzed directly (bottom) or after immunoprecipitation with anti-Bag6 or anti-SRP54. Diagram of results; Bag6 complex is green, SRP is blue. (c) As in panel b, but using TR-β and RT-β in the top and bottom panels, respectively. (d) The indicated constructs were translated in vitro, immunoaffinity purified via the N-terminus, and immunoblotted with anti-TRC40 or anti-Ubl4A (to detect the Bag6 complex). Autoradiograph shows equal recovery of the translated substrates.

Fig. 4. Maximal ubiquitination of MLPs requires Bag6

(a) Various constructs were assayed for ubiquitination in lysates containing or lacking Bag6. The ubiquitin gels of ΔSSΔGPI and β(3R) were exposed ~3 times longer than PrP and Sec61β. (b) Bag6-depleted lysates were replenished with recombinant Bag6 (Sup. Fig. S16), ΔUbl-Bag6, or native Bag6 complex, and tested for ubiquitination of TR-β. Relative Bag6 levels are indicated. (c) TR-β interacts with recombinant Bag6 and ΔUbl-Bag6 by crosslinking. (d) The indicated PrP constructs were co-transfected with Bag6 complex, ΔUbl-Bag6 complex, or irrelevant plasmid (see Sup. Fig. S20) and detected by immunoblotting. One sample was treated with proteasome inhibitor (MG132) for 4 h. Loading control is also shown. (e) Effect of ΔUbl-Bag6 complex on wild type PrP and Prl-PrP. Unglycosylated precursor PrP is preferentially stabilized by either ΔUbl-Bag6 complex overexpression or proteasome inhibition. (f) Model: the Bag6 complex captures ribosomally released hydrophobic proteins and triages them between post-translational targeting (TA proteins) and ubiquitination.