Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.

Maltose and maltotriose transport into ale and lager brewer's yeast strains

Maltose and maltotriose are the two most abundant sugars in brewer s wort, and thus brewer s yeast s ability to utilize them efficiently is of major importance in the brewing process. The increasing tendency to utilize high and very-high-gravity worts containing increased concentrations of maltose and maltotriose renders the need for efficient transport of these sugars even more pronounced. Residual maltose and especially maltotriose are quite often present especially after high and very-high-gravity fermentations. Sugar uptake capacity has been shown to be the rate limiting factor for maltose and maltotriose utilization. The main aim of the present study was to find novel ways to improve maltose and maltotriose utilization during the main fermentation. Maltose and maltotriose uptake characteristics of several ale and lager strains were studied. Genotype determination of the genes needed for maltose and maltotriose utilization was performed. Maltose uptake inhibition studies were performed to reveal the dominant transporter types actually functioning in each of the strains. Temperature-dependence of maltose transport was studied for ale and for lager strains as well as for each of the single sugar transporter proteins Agt1p, Malx1p and Mtt1p. The AGT1 promoter regions of one ale and two lager strains were sequenced by chromosome walking and the promoter elements were searched for using computational methods. The results showed that ale and lager strains predominantly use different maltose and maltotriose transporter types for maltose and maltotriose uptake. Agt1 transporter was found to be the dominant maltose/maltotriose transporter in the ale strains whereas Malx1 and Mtt1- type transporters dominated in the lager strains. All lager strains studied were found to possess a non-functional Agt1 transporter. The ale strains were observed to be more sensitive to temperature decrease in their maltose uptake compared to the lager strains. Single transporters were observed to differ in their sensitivity to temperature decrease and their temperature-dependence was shown to decrease in the order Agt1≥Malx1>Mtt1. The different temperature-dependence between the ale and lager strains was observed to be due to the different dominant maltose/maltotriose transporters ale and lager strains possessed. The AGT1 promoter regions of ale and lager strains were found to differ markedly from the corresponding regions of laboratory strains. The ale strain was found to possess an extra MAL-activator binding site compared to the lager strains. Improved maltose and maltotriose uptake capacity was obtained with a modified lager strain where the AGT1 gene was repaired and put under the control of a strong promoter. Modified strains fermented wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. Significant savings in the main fermentation time were obtained when modified strains were used. In high-gravity wort fermentations 8 20% and in very-high-gravity wort fermentations even 11 37% time savings were obtained. These are economically significant changes and would cause a marked increase in annual output from the same-size of brewhouse and fermentor facilities.

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Transmembrane domains participate in functions of integral membrane proteins

Integral membrane proteins have one or more transmembrane a-helical domains and carry out a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. These transmembrane domains are not mere structural units anchoring the protein to the lipid bilayer but seem to-contribute in the overall activity. Recent findings in support of this are described using some typical examples-LDL receptor, growth factor receptor tyrosine kinase, HMG-CoA reductase, F-0-ATPase and adrenergic receptors. The trends in research indicate that these transmembrane domains participate in a variety of ways such as a linker, a transducer or an exchanger in the overall functions of these proteins in transfer of materials, energy and signals.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, Delta GRT1 Delta GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Delta GRT1 Delta GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Delta GRT1 Delta GRT2 cells appear less adhesive than those from the wild type.

The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.