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The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.

The slow delayed rectifier potassium current, I Ks , conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, I Ks is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic up-regulation of I Ks requires recruitment of PKA holoenzyme (two regulatory – RI or RII – and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of I Ks in the absence of AKAP9. Targeted recruitment of endogenous Cα to E1-YFP using a GFP/YFP nanobody (nano) fused to RIIα enabled acute cAMP-mediated enhancement of I Ks , reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited I Ks by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed that distinct phosphorylation sites are modified by Cα targeted to Q1-YFP compared to free Cα. Thus, functional outcomes of synthetically recruited PKA on I Ks regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of I Ks by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.

Polyubiquitin chain diversity generates a ‘ubiquitin code’ that universally regulates protein abundance, localization, and function. Functions of polyubiquitin diversity are mostly unknown, with lack of progress due to an inability to selectively tune protein polyubiquitin linkages in live cells. We develop linkage-selective engineered deubiquitinases (enDUBs) by fusing linkage-selective DUB catalytic domains to GFP-targeted nanobody and use them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. YFP-KCNQ1 in HEK293 cells has polyubiquitin chains with K48/K63 linkages dominant. EnDUBs yield unique effects on channel surface abundance with a pattern indicating: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling; and K48 is necessary for forward trafficking. EnDUB effects differ in cardiomyocytes and on KCNQ1 disease mutants, emphasizing ubiquitin code mutability. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 abundance and subcellular localization and introduce linkage-selective enDUBs as potent tools to demystify the polyubiquitin code. Polyubiquitin chains form a “ubiquitin code” to control protein functions. Here, the authors develop linkage-selective enDUBs to study this in live cells. Applied to Kv7.1 ion channel, enDUBs reveal distinct roles for K11, K29/33, K48, and K63 chains in regulating channel abundance and trafficking.

Targeted protein degradation/downregulation (TPD/TPDR) is a disruptive paradigm for developing therapeutics. <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit ion channels has not been demonstrated. NEDD4-2 E3 ligase regulates myriad ion channels, but its utility for TPD/TPDR is uncertain due to complex regulatory mechanisms. Here, we identify a nanobody that binds NEDD4-2 HECT domain without disrupting catalysis sites as revealed by cryo-electron microscopy and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels (Ca V 2.2; KCNQ1; and epithelial Na + channel, ENaC, with a Liddle syndrome mutation) using divalent nanobodies (DiVas) strongly suppresses their surface density and function. Global proteomics indicates DiVa recruitment of endogenous NEDD4-2 to KCNQ1-YFP yields dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of NEDD4-2 recruitment for TPD/TPDR, validate ion channels as susceptible to this modality, and introduce a general method to generate ion channel inhibitors. Researchers develop a new way to selectively remove ion channel proteins by recruiting the body’s own NEDD4-2 enzyme using custom nanobodies, offering a precise and general strategy for future drug development.

Polyubiquitin chain diversity generates a 'ubiquitin code' that universally regulates protein abundance, localization, and function. Functions of polyubiquitin diversity are mostly unknown, with lack of progress due to an inability to selectively tune protein polyubiquitin linkages in live cells. We develop linkage-selective engineered deubiquitinases (enDUBs) by fusing linkage-selective DUB catalytic domains to GFP-targeted nanobody and use them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. YFP-KCNQ1 in HEK293 cells has polyubiquitin chains with K48/K63 linkages dominant. EnDUBs yield unique effects on channel surface abundance with a pattern indicating: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling; and K48 is necessary for forward trafficking. EnDUB effects differ in cardiomyocytes and on KCNQ1 disease mutants, emphasizing ubiquitin code mutability. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 abundance and subcellular localization and introduce linkage-selective enDUBs as potent tools to demystify the polyubiquitin code.

The past several decades have witnessed tremendous growth in the protein targeting, transport and translocation field. Major advances were made during this time period. Now the molecular details of the targeting factors, receptors and the membrane channels that were envisioned in Blobel’s Signal Hypothesis in the 1970s have been revealed by powerful structural methods. It is evident that there is a myriad of cytosolic and membrane associated systems that accurately sort and target newly synthesized proteins to their correct membrane translocases for membrane insertion or protein translocation. Here we will describe the common principles for protein transport in prokaryotes and eukaryotes.

Chapter 1 of this dissertation reviews existing knowledge of the areas of protein translocation and membrane insertion. The core machineries involved in membrane protein biogenesis are remarkably conserved. Proteins that fold within the cell prior to export are translocated by the Tat system. Canonical substrates of this pathway possess signal sequences with a twin-arginine motif which interacts with the TatABC membrane translocon complex to facilitate substrate translocation. The majority of the secreted and membrane proteins are translocated by the Sec machinery in an unfolded state. It consists of the SecY, SecE and SecG proteins which form an hour-glass shaped channel with a lateral gate opening into the membrane. Substrate targeting to the Sec translocon occurs either post-translationally or co-translationally. Most exported proteins in E. coli are post-translationally targeted by the SecA/B pathway. The SecB is a molecular chaperone that delivers a subset of substrate proteins in an unfolded state to SecA. The SecA motor ATPase powers the movement of substrates through the SecYEG channel. Co-translational targeting typically involves the association of the translating ribosome with the translocase directly, or with the translocase after delivery by the signal-recognition particle (SRP) and its receptor (SR). Certain substrates of the SRP pathway are targeted to another translocon, YidC. YidC plays a pivotal role in the membrane integration, folding and assembly of a subset of proteins including energy-transducing and respiratory complexes. It functions both autonomously and in concert with the SecYEG channel in bacteria. The YidC family of proteins are widely conserved in all domains of life with new members recently identified in the eukaryotic ER membrane. Bacterial and organellar members share the conserved 5 TM core which forms a unique hydrophilic cavity in the inner leaflet of the bilayer accessible from the cytoplasm and the lipid phase. The work presented here investigates the pathway-determining factors for amino-terminal translocation in E. coli. In addition, the conserved function of the YidC family of proteins to insert a single-spanning protein into the membrane was explored using biophysical methods.Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion and assembly of inner membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In Chapter 2, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36 residue N-tail of 2Pf3-Lep translocates independent of YidC-Sec. This is the longest N-tail region that is translocated by this pathway. We confirmed this using a newly constructed YidC-Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC-Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted, however an MBP-2Pf3-Lep fusion protein could be translocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC-Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in E. coli.The YidC family of proteins share structural homology and engage in the process of membrane protein biogenesis of various cellular and organellar membranes. Chapter 4 explores the functional conservation amongst the eukaryotic YidC homologs by testing the insertion of the YidC-only model substrate Pf3 coat protein. Fluorescence correlation spectroscopy technique was utilized to follow the insertion process at the single-molecule level in vitro. The thylakoid membrane of chloroplasts contains 2 YidC-paralogs: Alb3 and Alb4. Although both have distinct set of substrates in the chloroplasts, we found that they can insert Pf3 coat substrate with comparable efficiencies to YidC. This is in agreement with the fact that these insertases can complement YidC in E. coli. We also tested the mitochondrial homolog Oxa1 and the newly proposed ER-resident member Get1. Oxa1 is a bonafide member of the YidC family; both YidC and Oxa1 can complement one another. However, experimental evidence is lacking to confirm the placement of Get1 in the YidC family. Interestingly, both Oxa1 and Get1 can insert Pf3 coat protein into reconstituted proteoliposomes. This suggests that Get1 and the other homologs tested are functionally conserved. This study provides fundamental information about the evolutionarily conserved role of YidC in membrane protein biogenesis.

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite- as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and Ca 2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.