RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.     

Syntaxin 16 Binds to Cystic Fibrosis Transmembrane Conductance Regulator and Regulates Its Membrane Trafficking in Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a key membrane protein in the complex network of epithelial ion transporters regulating epithelial permeability. Syntaxins are one of the major determinants in the intracellular trafficking and membrane targeting of secretory proteins. In the present study we demonstrate the biochemical and functional association between CFTR and syntaxin 16 (STX16) that mediates vesicle transport within the early/late endosomes and trans-Golgi network. Immunoprecipitation experiments in rat colon and T84 human colonic epithelial cells indicate that STX16 associates with CFTR. Further analyses using the domain-specific pulldown assay reveal that the helix domain of STX16 directly interacts with the N-terminal region of CFTR. Immunostainings in rat colon and T84 cells show that CFTR and STX16 highly co-localize at the apical and subapical regions of epithelial cells. Interestingly, CFTR-associated chloride current was reduced by the knockdown of STX16 expression in T84 cells. Surface biotinylation and recycling assays indicate that the reduction in CFTR chloride current is due to decreased CFTR expression on the plasma membrane. These results suggest that STX16 mediates recycling of CFTR and constitutes an important component of CFTR trafficking machinery in intestinal epithelial cells.     

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl⁻ secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na⁺ absorption. The mechanistic link between missing CFTR and increased Na⁺ absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na⁺ channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na⁺ absorption.     

Ubiquitin E3 Ligase A20 Facilitates Processing Microbial Product in Nasal Epithelial Cells

Microbial products play a role in the pathogenesis of allergic diseases; ubiquitin E3 ligase A20 (A20) is an important molecule in regulating inflammation in the body. The present study aims to elucidate the role of A20 in processing the absorbed microbial products in nasal epithelial cells. Human nasal mucosal specimens were collected from patients with or without chronic rhinitis and analyzed by immunohistochemistry. Human nasal epithelial cell line, RPMI2650 cell, was employed to assess the role of A20 in processing the absorbed staphylococcal enterotoxin B (SEB). The RPMI2650 cells absorbed SEB in the culture. The increase in A20 was observed in RPMI2650 cells in parallel to the absorption of SEB. A20 is a critical molecule in the degradation of SEB in the nasal epithelial cells by promoting the tethering of endosomes and lysosomes. A20 plays a critical role in processing of the absorbed SEB in nasal epithelial cells.     

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N⁶-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N⁶-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻ channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest β strands (β_c5 and β_c6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the β_c5-β_c6 hairpin, within the β_c5 strand (H620Q), within the β-turn linking the two β strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the β_c6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.     

Folding and Rescue of a Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Mutant Identified Using Human-Murine Chimeric Proteins

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys⁵⁸⁴ interacts with residue Leu⁵⁸¹ in human CFTR Glu⁵⁸⁴ interacts with Phe⁵⁸¹ in mouse CFTR. Introduction of the murine residue (Phe⁵⁸¹) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.     

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.     

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser⁷³⁷ in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl⁻ secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser⁷³⁷ mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl⁻ channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl⁻ channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.     

Converting Nonhydrolyzable Nucleotides to Strong Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Agonists by Gain of Function (GOF) Mutations

Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5′-O-(thiotriphosphate) (ATPγS), adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), and guanosine 5′-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τ_deac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.     

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg³⁵²-Asp⁹⁹³ and Arg³⁴⁷-Asp⁹²⁴, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg³⁴⁷ not only interacts with Asp⁹²⁴ but also interacts with Asp⁹⁹³. The tripartite interaction Arg³⁴⁷-Asp⁹²⁴-Asp⁹⁹³ mainly contributes to maintaining a stable s2 open subconductance state. The Arg³⁵²-Asp⁹⁹³ salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg³⁵² and Asp⁹⁹³, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.     

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.     

Smad Ubiquitylation Regulatory Factor 1/2 (Smurf1/2) Promotes p53 Degradation by Stabilizing the E3 Ligase MDM2

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.