Direct Contact Sites Research Articles

Analysis of a Super-Complex at Contact Sites Between Mitochondria and Plastids.

Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures regulated in response to stress. For example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (Mitochondrial Transmembrane Lipoprotein) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex.

Monkeypox: Cutaneous clues to clinical diagnosis

Monkeypox: Cutaneous clues to clinical diagnosis

XAV-19, a Swine Glyco-Humanized Polyclonal Antibody Against SARS-CoV-2 Spike Receptor-Binding Domain, Targets Multiple Epitopes and Broadly Neutralizes Variants.

Amino acid substitutions and deletions in the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBMs), in direct contact sites with the angiotensin converting enzyme-2 (ACE-2). Therefore, in Spike/ACE-2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study on Vero E6 cells conducted over 1 month, no mutation was associated with the addition of increasing doses of XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE-2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (COVID-19) including the different variants of concern identified so far.

Cal'MAM'ity at the Endoplasmic Reticulum-Mitochondrial Interface: A Potential Therapeutic Target for Neurodegeneration and Human Immunodeficiency Virus-Associated Neurocognitive Disorders.

The endoplasmic reticulum (ER) is a multifunctional organelle and serves as the primary site for intracellular calcium storage, lipid biogenesis, protein synthesis, and quality control. Mitochondria are responsible for producing the majority of cellular energy required for cell survival and function and are integral for many metabolic and signaling processes. Mitochondria-associated ER membranes (MAMs) are direct contact sites between the ER and mitochondria that serve as platforms to coordinate fundamental cellular processes such as mitochondrial dynamics and bioenergetics, calcium and lipid homeostasis, autophagy, apoptosis, inflammation, and intracellular stress responses. Given the importance of MAM-mediated mechanisms in regulating cellular fate and function, MAMs are now known as key molecular and cellular hubs underlying disease pathology. Notably, neurons are uniquely susceptible to mitochondrial dysfunction and intracellular stress, which highlights the importance of MAMs as potential targets to manipulate MAM-associated mechanisms. However, whether altered MAM communication and connectivity are causative agents or compensatory mechanisms in disease development and progression remains elusive. Regardless, exploration is warranted to determine if MAMs are therapeutically targetable to combat neurodegeneration. Here, we review key MAM interactions and proteins both in vitro and in vivo models of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. We further discuss implications of MAMs in HIV-associated neurocognitive disorders (HAND), as MAMs have not yet been explored in this neuropathology. These perspectives specifically focus on mitochondrial dysfunction, calcium dysregulation and ER stress as notable MAM-mediated mechanisms underlying HAND pathology. Finally, we discuss potential targets to manipulate MAM function as a therapeutic intervention against neurodegeneration. Future investigations are warranted to better understand the interplay and therapeutic application of MAMs in glial dysfunction and neurotoxicity.

Systems modeling predicts that mitochondria ER contact sites regulate the postsynaptic energy landscape

Spatiotemporal compartmentation of calcium dynamics is critical for neuronal function, particularly in postsynaptic spines. This exquisite level of Ca2+ compartmentalization is achieved through the storage and release of Ca2+ from various intracellular organelles particularly the endoplasmic reticulum (ER) and the mitochondria. Mitochondria and ER are established storage organelles controlling Ca2+ dynamics in neurons. Mitochondria also generate a majority of energy used within postsynaptic spines to support the downstream events associated with neuronal stimulus. Recently, high resolution microscopy has unveiled direct contact sites between the ER and the mitochondria (MERCs), which directly channel Ca2+ release from the ER into the mitochondrial membrane. In this study, we develop a computational 3D reaction-diffusion model to investigate the role of MERCs in regulating Ca2+ and ATP dynamics. This spatiotemporal model accounts for Ca2+ oscillations initiated by glutamate stimulus of metabotropic and ionotropic glutamate receptors and Ca2+ changes in four different compartments: cytosol, ER, mitochondria, and the MERC microdomain. Our simulations predict that the organization of these organelles and inter-organellar contact sites play a key role in modulating Ca2+ and ATP dynamics.We further show that the crosstalk between geometry (mitochondria and MERC) and metabolic parameters (cytosolic ATP hydrolysis, ATP generation) influences the neuronal energy state. Our findings shed light on the importance of organelle interactions in predicting Ca2+ dynamics in synaptic signaling. Overall, our model predicts that a combination of MERC linkage and mitochondria size is necessary for optimal ATP production in the cytosol.

ER‐associated Regulation of Astrocyte Mitochondrial Function during METH Exposure and HIV‐1 Infection

Astrocytes are key regulators of central nervous system (CNS) health and neuronal function. Astrocyte mitochondrial dysfunction, such as induced by METH and HIV-1, threatens the provision of essential metabolic and antioxidant support to neurons. Thus, delineating regulatory pathways that can be targeted to prevent aberrant mitochondria homeostasis in astrocytes will be imperative for ensuring neuronal fitness/survival against CNS pathologies. Direct contact sites between the endoplasmic reticulum (ER) and the mitochondria, termed mitochondrial associated membranes (MAMs), are central hubs for regulating several cellular processes required for homeostasis, including mitochondrial metabolic activity. In fact, the transfer of Ca2+ from the ER to mitochondria is essential for mitochondrial bioenergetics. Recent investigations have also identified unique, yet ill-defined contributions of the three unfolded protein response (UPR) arms, beyond their classical ER stress functions, in regulating MAM tethering and/or signaling. Briefly, protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been determined as a key regulator for MAM tethering, inositol-requiring kinase 1 (IRE1α) is implicated in regulating MAM-mediated Ca2+ transfer, and activating transcription factor 6 (ATF6) is suspected to participate in MAM formation as it is known to mediate ER elongation and lipid homeostasis. However, these regulatory mechanisms have not yet been fully elucidated. The current investigation examined changes in astrocyte mitochondrial function, ER stress, Ca2+ signaling, and the regulation of MAMs in response to METH exposure and HIV-1 infection. Then, we explored the role of ER-associated mechanisms in regulating astrocyte mitochondrial function. The effects of METH treatment were evaluated in both acute and chronic paradigms while responses to chronic HIV-1 infection was examined using a pseudotyped HIV-1 virion able to infect astrocytes. Astrocyte metabolic status was determined using Seahorse extracellular flux analyzer for a real-time assessment of cellular metabolism in addition to quantifying the expression profiles of essential metabolites. Changes in the expression of unfolded protein response (UPR) and MAM mediators were determined using RT-PCR and protein expression assays. Finally, pharmacological inhibition of the UPR pathways were used to delineate the ER-associated regulatory mechanisms mediating the changes in mitochondria bioenergetics. Our studies demonstrate increased astrocyte metabolic capacity in response to chronic METH exposure and HIV-1 infection which corresponded to increased expression of UPR/MAM mediators. Moreover, pharmacological inhibition of specifically IRE1α impaired astrocyte mitochondrial activity. These findings illustrate the importance of ER-mitochondria communication in regulating astrocyte mitochondrial function and identifies a possible mechanism to manipulate the metabolic and antioxidant coupling between astrocytes and neurons during METH/HIV-1 pathogenesis.

Nickel Allergy: Epidemiology, Pathomechanism, Clinical Patterns, Treatment and Prevention Programs.

Nickel is the most common cause of contact allergy in the general population and the most frequently detected allergen in patients patch tested for suspected allergic contact dermatitis (ACD). ACD from nickel is a typical type IV hypersensitivity. Nickel allergy is mostly caused by nonoccupational exposure, such as jewelry and clothing decorations, metal tools, medical devices (mainly orthopedic and orthodontic implants, cardiovascular prosthesis), eyeglasses, utensils, keys, pigment for paint, cosmetics, and food (mainly legumes, chocolate, salmon, peanuts). Occupational exposure can involve several workers (mechanics, metalworkers, platers, hairdressers, jewelers, workers in the constructions and electronic industries), classically involving hands and forearms. The classic clinical pattern of ACD caused by nickel is characterized by eczematous dermatitis involving the sites of direct contact with the metal. Non-eczematous-patterns are reported, including lichenoid dermatitis, granuloma annulare, vitiligo-like lesions, dyshidrosiform dermatitis, and vasculitis. In the case of systemic exposure to nickel, sensitized patients could develop systemic contact dermatitis. Patch testing represents the gold standard for the diagnosis of ACD from nickel. Treatment includes avoidance of contact with products containing nickel and the patient's education about the possible use of alternative products. A recent EU nickel directive, regulating the content and release of nickel from products, has caused a decrease of nickel contact allergy in some European countries. Nickel allergy is a relevant issue of public health with significant personal, social, and economic impact. This review summarizes epidemiology, pathomechanism, clinical patterns, treatment, and prevention programs.

Electronic structure of the clean interface between single crystal CH3NH3PbI3 and an organic hole transporting material spiro-OMeTAD

Methylammonium lead triiodide (CH3NH3PbI3) is a fundamental material used for prototypical perovskite solar cells. The electronic properties of the interface between CH3NH3PbI3 and hole transporting materials play a crucial role in the efficient performance of these solar cells. However, the intrinsic characteristics of the interfaces where these materials directly come into contact with each other have not yet been defined since previous studies were performed using polycrystalline thin films of CH3NH3PbI3, which were confirmed to contain a considerable amount of impurities. In this study, x-ray photoelectron spectroscopy and ultraviolet photoelectron spectroscopy were conducted to determine the interfacial electronic structure between CH3NH3PbI3 and 2,2′,7,7′-tetrakis-(N,N-di-p-methoxyphenylamino)-9,9′-spirobifluorene (spiro-OMeTAD) on the clean interface formed on the impurity-free single crystal surface of CH3NH3PbI3. Spontaneous hole injection from CH3NH3PbI3 to spiro-OMeTAD occurred at the direct contact sites between these materials, a phenomenon that was confirmed to be hindered by the presence of impurities at the interface.

Role of Helix 8 in Dopamine Receptor Signaling.

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.

Cholesterol Crystals in Hepatocyte Lipid Droplets Are Strongly Associated With Human Nonalcoholic Steatohepatitis.

It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs. Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis (P < 0.05). We were unable to identify significant differences in the expression of genes in liver tissue related to cholesterol homeostasis or LD proteins between patients with fibrosing NASH and isolated steatosis. Human hepatoma cell line (HepG2) cells were supplemented with low‐density lipoprotein (LDL)‐cholesterol and oleic acid to develop large LDs, similar to those observed in patients with NASH. Fluorescent markers were used to track the uptake and intracellular trafficking of LDL‐cholesterol. LDL‐cholesterol was taken up by HepG2 cells and transported through the endosomal‐lysosomal compartment directly to LDs, suggesting direct contact sites between late endosomes and LDs. Exposure of HepG2 cells to LDL‐cholesterol resulted in a high concentration of cholesterol and cholesterol crystallization in LDs. Conclusion: Excess cholesterol is stored in the liver primarily within hepatocyte LDs where it can crystallize. Our findings are best explained by direct transport of cholesterol from late endosomes/lysosomes to LDs in hepatocytes. We found a strong association between the presence of LD cholesterol crystals and the development of fibrosing NASH in humans, suggesting a causal relationship.

Analysis of the MTL Supercomplex at Contact Sites Between Mitochondria and Plastids.

Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures that are modified in response to stress. As example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (mitochondrial transmembrane lipoprotein complex) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex.

The hardness and chemical changes in demineralized primary dentin treated by fluoride and glass ionomer cement

Abstract Background Fluoride plays an important role in the control of dental caries. Aim To evaluate the chemical exchange between restoration of glass ionomer cement of high viscosity (GIC) and primary dentin with application of sodium fluoride (NaF) 2% through changes in hardness from uptake of calcium, phosphate and fluoride. Material and method Class I cavities were prepared in 40 sound primary molars, and the sample was divided into two groups (n=20) according to dentin condition: sound (1) and demineralized (2). Sub-groups (n=10) were formed to investigate the isolated action of the GIC or the association with NaF (F). This in vitro study examined the chemical exchange under two conditions, sound and demineralized dentin (pH cycling), to simulate the occurrence of mineral loss for the caries lesion. G1 and G2 received GIC restoration only; groups G1F and G2F received NaF before GIC restoration. The specimens were prepared for Knoop hardness test and micro-Raman spectroscopy. A two-way ANOVA test (α = 0.05) was used for statistical analysis. Micro-Raman data were qualitatively described. Result Increased hardness was observed in all the sites of direct contact with GIC in sound and demineralized dentin for all groups (p&lt;0.001); no difference was observed in microhardness after application of NaF (p&gt;0.05). In the evaluation of micro-Raman, direct contact between GIC and dentin for sound and demineralized dentin resulted in increased peaks of phosphate. Conclusion The exchange between GIC and demineralized dentin may induce changes of mechanical properties of the substrate, and uptake of mineral ions (phosphate) occurs without the influence of NaF.

Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus.

BackgroundCorals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.ResultsTag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.ConclusionsSimilarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1540-2) contains supplementary material, which is available to authorized users.

Communicating by touch--neurons are not alone.

Long-distance cell-cell communication is essential for organ development and function. Whereas neurons communicate at long distances by transferring signals at sites of direct contact (i.e., at synapses), it has been presumed that the only way other cell types signal is by dispersing signals through extracellular fluid--indirectly. Recent evidence from Drosophila suggests that non-neuronal cells also exchange signaling proteins at sites of direct contact, even when long distances separate the cells. We review here contact-mediated signaling in neurons and discuss how this signaling mechanism is shared by other cell types.

Cytonemes extend their reach

Nature advance online publication April282013; doi:10.1038/nature12157 Cytonemes are specialized filopodia, first described in the Drosophila wing imaginal disc, that were proposed to mediate long distance signalling during development. A recent report from Barna and colleagues (Sanders et al, 2013) published in Nature shows that cytonemes in the chick limb bud carry SHH signalling proteins out to signal receiving cells, thus highlighting their evolutionarily conserved roles in cell–cell communication.

Delineating the regions of human transferrin involved in interactions with transferrin binding protein B from Neisseria meningitidis

Pathogenic bacteria in the Neisseriaceae possess a surface receptor mediating iron acquisition from human transferrin (hTf) that consists of a transmembrane iron transporter (TbpA) and a surface-exposed lipoprotein (TbpB). In this study, we used hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS) to elucidate the effects on hTf by interaction with TbpB or derivatives of TbpB. An overall conserved interaction was observed between hTf and full-length or N-lobe TbpB from Neisseria meningitidis strains B16B6 or M982 that represent two distinct subtypes of TbpB. Changes were observed exclusively in the C-lobe of hTf and were caused by the interaction with the N-lobe of TbpB. Regions localized to the 'lip' of the C1 and C2 domains that flank the interdomain cleft represent sites of direct contact with TbpB whereas the peptides within the interdomain cleft that encompass iron binding ligands are inaccessible in the closed (holo) conformation. Although substantial domain separation upon binding TbpB cannot be excluded by the H/DX-MS data, the preferred model of interaction involves binding hTf C-lobe in the closed conformation. Alternate explanations are provided for the substantial protection from deuteration of the peptides encompassing iron binding ligands within the interdomain cleft but cannot be differentiated by the H/DX-MS data.

CO oxidation on non-alloyed Pt and Ru electrocatalysts prepared by the polygonal barrel-sputtering method

The CO oxidation on non-alloyed Pt and Ru electrocatalysts prepared by the polygonal barrel-sputtering method was investigated. Samples were prepared by sputtering Pt and Ru separately at room temperature. From the X-ray diffraction (XRD) measurement of the sample prepared on a SiO 2 glass plate, it was found that the sputtered metals are non-alloyed. Subsequently, the non-alloyed Pt and Ru electrocatalysts were prepared by the polygonal barrel-sputtering method using carbon powder as a support. The XRD patterns of these samples showed a single and very broad peak supporting the hypothesis of the non-alloyed Pt and Ru. No separate Ru reflections were visible, which could be attributed to Ru particle sizes smaller than 4 nm, as obtained by transmission electron microscopy (TEM). CO oxidation on the non-alloyed Pt and Ru electrocatalysts were evaluated by CO stripping voltammetry. This measurement revealed that the lower peak potential of CO oxidation varies, depending on the Pt content and the sputtering order. In addition, it was assumed that the CO oxidation reaction site for non-alloyed Pt and Ru electrocatalyst has a limited area including direct contact sites between the Pt and Ru particles.

Assembly of GABAAreceptors (Review)

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by gamma-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA(A) receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA(A) receptor subtypes in the brain. The majority of GABA(A) receptors seems to be composed of two alpha, two beta and one gamma subunit and the occurrence of a defined subunit stoichiometry and arrangement in alphabetagamma receptors strongly indicates that assembly of GABA(A) receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA(A) receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA(A) receptors are discussed in the light of recent publications.

Mapping an interface of SecY (PrlA) and SecE (PrlG) by using synthetic phenotypes and in vivo cross-linking.

SecY and SecE are integral cytoplasmic membrane proteins that form an essential part of the protein translocation machinery in Escherichia coli. Sites of direct contact between these two proteins have been suggested by the allele-specific synthetic phenotypes exhibited by pairwise combinations of prlA and prlG signal sequence suppressor mutations in these genes. We have introduced cysteine residues within the first periplasmic loop of SecY and the second periplasmic loop of SecE, at a specific pair of positions identified by this genetic interaction. The expression of the cysteine mutant pair results in a dominant lethal phenotype that requires the presence of DsbA, which catalyzes the formation of disulfide bonds. A reducible SecY-SecE complex is also observed, demonstrating that these amino acids must be sufficiently proximal to form a disulfide bond. The use of cysteine-scanning mutagenesis enabled a second contact site to be discovered. Together, these two points of contact allow the modeling of a limited region of quaternary structure, establishing the first characterized site of interaction between these two proteins. This study proves that actual points of protein-protein contact can be identified by using synthetic phenotypes.

Fluorescence-based protein footprinting using histidine-tagged protein.

We describe a procedure for protein footprinting to identify the region(s) of a protein that interacts with a ligand. The method utilized the affinity of a stretch of histidine residues cloned into the protein to metal-chelated resin. After limited protease digestion, the histidine-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled digestion products on a denaturing polyacrylamide gel and visualizing the peptides using a FluorImager provided a way to identify the protease target sites that were protected from digestion because of interaction with DNA. The protection experiments would be applicable not only to detect direct contact sites but also sites allosterically altered by ligand binding.