Small-molecule displacement of a cryptic degron causes conditional protein degradation

The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.     

RNF121 Inhibits Angiogenic Growth Factor Signaling by Restricting Cell Surface Expression of VEGFR‐2

Ligand stimulation promotes downregulation of RTKs, a mechanism by which RTKs, through the ubiquitination pathway are removed from the cell surface, causing a temporary termination of RTK signaling. The molecular mechanisms governing RTK trafficking and maturation in the endoplasmic reticulum (ER)/Golgi compartments are poorly understood. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is a prototypic RTK that plays a critical role in physiologic and pathologic angiogenesis. Here we demonstrate that Ring Finger Protein 121 (RNF121), an ER ubiquitin E3 ligase, is expressed in endothelial cells and regulates maturation of VEGFR‐2. RNF121 recognizes newly synthesized VEGFR‐2 in the ER and controls its trafficking and maturation. Over‐expression of RNF121 promoted ubiquitination of VEGFR‐2, inhibited its maturation and resulted a significantly reduced VEGFR‐2 presence at the cell surface. Conversely, the shRNA‐mediated knockdown of RNF121 in primary endothelial cells reduced VEGFR‐2 ubiquitination and increased its cell surface level. The RING Finger domain of RNF121 is required for its activity toward VEGFR‐2, as its deletion significantly reduced the effect of RNF121 on VEGFR‐2. Additionally, RNF121 inhibited VEGF‐induced endothelial cell proliferation and angiogenesis. Taken together, these data identify RNF121 as a key determinant of angiogenic signaling that restricts VEGFR‐2 cell surface presence and its angiogenic signaling.      

Arrestin-Mediated Endocytosis of Yeast Plasma Membrane Transporters

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.     

A cytosolic domain of the yeast Zrt1 zinc transporter is required for its post-translational inactivation in response to zinc and cadmium

Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells. Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition. Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals. We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis. During the transition from zinc-limiting to zinc-replete growth conditions (i.e. zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole. To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane. This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking. Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells.     

Serine-threonine ubiquitination mediates downregulation of BST-2/tetherin and relief of restricted virion release by HIV-1 Vpu

The HIV-1 protein Vpu counteracts the antiviral activity of the innate restriction factor BST-2/tetherin by a mechanism that partly depends on its interaction with β-TrCP, a substrate adaptor for an SCF (Skp-Cullin 1-F box) E3 ubiquitin ligase complex. This suggests that Vpu stimulates the ubiquitination of BST-2 and that this underlies the relief of restriction. Here, we show that Vpu stimulates ubiquitination of BST-2. Mutation of all potential ubiquitination sites in the cytoplasmic domain of BST-2, including lysines, cysteines, serines, and threonines, abrogates Vpu-mediated ubiquitination. However, a serine-threonine-serine sequence specifically mediates the downregulation of BST-2 from the cell surface and the optimal relief of restricted virion release. Serine-threonine ubiquitination of BST-2 is likely part of the mechanism by which Vpu counteracts innate defenses.     

Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms

The function of many receptors and transport proteins that reside at the surface of the cell is regulated by endocytosis and postendocytic trafficking. Modification of receptors and transporters by ubiquitin conjugation has recently emerged as the major regulatory mechanism of internalization and intracellular sorting of these membrane proteins. This review will describe recent advances in elucidating the mechanisms of ubiquitination of mammalian receptors and transporters using two examples: the receptor for epidermal growth factor and the dopamine transporter. How ubiquitination controls the endocytosis and turnover of these proteins will be also discussed.     

A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity

Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in lon which affected its substrate specificity. The most informative lon mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within lon leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.     

An intrinsic network of polar interactions is responsible for binding of UL49.5 C-degron by the CRL2KLHDC3 ubiquitin ligase

Bovine herpesvirus type 1 (BoHV-1) is a pathogen of cattle responsible for infectious bovine rhinotracheitis. The BoHV-1 UL49.5 is a transmembrane protein that binds to the transporter associated with antigen processing (TAP) and downregulates cell surface expression of the antigenic peptide complexes with the major histocompatibility complex class I (MHC-I). KLHDC3 is a kelch domain-containing protein 3 and a substrate receptor of a cullin2-RING (CRL2) E3 ubiquitin ligase. Recently, it has been identified that CRL2^KLHDC3 is responsible for UL49.5-triggered TAP degradation via a C-degron pathway and the presence of the degron sequence does not lead to the degradation of UL49.5 itself. The molecular modeling of KLHDC3 in complexes with four UL49.5 C-terminal decapeptides (one native protein and three mutants) revealed their activity to be closely correlated with the conformation which they adopt in KLHDC3 binding cleft. To analyze the interaction between UL49.5 and KLHDC3 in detail, in this work a total of 3.6 μs long molecular dynamics simulations have been performed. The complete UL49.5-KLHDC3 complexes were embedded into the fully hydrated all-atom lipid membrane model with explicit water molecules. The network of polar interactions has been proposed to be responsible for the recognition and binding of the degron in KLHDC3. The interaction network within the binding pocket appeared to be very similar between two CRL2 substrate receptors: KLHDC3 and KLHDC2.     

Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner

Leishmania parasites experience variable nutrient levels as they cycle between the extracellular promastigote stage in the sandfly vector and the obligate intracellular amastigote stage in the mammalian host. Here we show that the surface expression of three Leishmania mexicana hexose and myo-inositol transporters is regulated in both a stage-specific and nutrient-dependent manner. GFP-chimeras of functionally active hexose transporters, LmGT2 and LmGT3, and the myo-inositol transporter, MIT, were primarily expressed in the cell body plasma membrane in rapidly dividing promastigote stages. However MIT-GFP was mostly rerouted to the multivesicular tubule (MVT)-lysosome when promastigotes reached stationary phase growth and all three nutrient transporters were targeted to the amastigote lysosome following transformation to in vitro differentiated or in vivo imaged amastigote stages. This stage-specific decrease in surface expression of GFP-tagged transporters correlated with decreased hexose or myo-inositol uptake in stationary phase promastigotes and amastigotes. The MVT-lysosme targeting of the MIT-GFP protein was reversed when promastigotes were deprived of myo-inositol, indicating that nutrient signals can override stage-specific changes in transporter distribution. The surface expression of the hexose and myo-inositol transporters was not regulated by interactions with the subpellicular cytoskeleton, as both classes of transporters associated with detergent-resistant membranes. LmGT3-GFP and MIT-GFP proteins C-terminally modified with mono-ubiquitin were constitutively transported to the MVT-lysosome, suggesting that ubiquitination may play a key role in regulating the subcellular distribution of these transporters and parasite adaptation to different nutrient conditions.     

A Substrate Binding Hinge Domain Is Critical for Transport-related Structural Changes of Organic Cation Transporter 1

Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.     

Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I

The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.     

Regulation of human organic anion transporter 4 by progesterone and protein kinase C in human placental BeWo cells

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the placenta. In the current study, we examined the regulation of hOAT4 by pregnancy-specific hormones progesterone (P(4)) and 17beta-estradiol (E(2)) and by protein kinase C (PKC) in human placental BeWo cells. P(4) induced a time- and concentration-dependent downregulation of hOAT4 transport activity, whereas E(2) had no effect on hOAT4 function. The downregulation of hOAT4 activity by P(4) mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased V(max) without significant change in K(m). Activation of PKC by phorbol 12,13-dibutyrate also resulted in an inhibition of hOAT4 activity through a decreased cell surface expression of the transporter. However, P(4)-induced downregulation of hOAT4 activity could not be prevented by treating hOAT4-expressing cells with the PKC inhibitor staurosporine. We concluded that both P(4) and activation of PKC inhibited hOAT4 activity through redistribution of the transporter from cell surface to the intracellular compartments. However, P(4) regulates hOAT4 activity by mechanisms independent of PKC pathway.     

Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.     

Recognition of a single transmembrane degron by sequential quality control checkpoints

To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the alpha-subunit of the T-cell antigen receptor (TCRalpha) into the alpha-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only approximately 50% of nascent mutant HA chains (HA(++)) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining approximately 50% of nascent HA(++) chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA(++) molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA(wt). These apparently native HA(++) molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.     

DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(βTrCP) E3 ubiquitin ligase and regulates survival and autophagy

DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCF(βTrCP) E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F box protein, βTrCP, via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by βTrCP. DEPTOR half-life is shortened by βTrCP but extended by a dominant-negative mutant of βTrCP, by RSK1/S6K1 inhibition, and by βTrCP degron site mutations. Biologically, DEPTOR accumulation upon βTrCP knockdown inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR accumulates upon glucose deprivation and mTOR inhibition to induce autophagy. Thus, βTrCP-DEPTOR-mTOR intertwine to regulate cell survival and autophagy.