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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.

SUBMITTER: Hessa T

PROVIDER: S-EPMC3150218 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Protein targeting and degradation are coupled for elimination of mislocalized proteins.

Hessa Tara T Sharma Ajay A Mariappan Malaiyalam M Eshleman Heather D HD Gutierrez Erik E Hegde Ramanujan S RS

Nature 20110710 7356

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 ribosome ...[more]

PMID: 21743475

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Project description:The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

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Protein targeting and degradation are coupled for elimination of mislocalized proteins.

Publications

Protein targeting and degradation are coupled for elimination of mislocalized proteins.

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