We thank Dr. R. Mart&#237;nez-de-Mena, M. M. Mu&#241;oz-Hernandez, A., Dr C. Jimenez and E. R. Mart&#237;nez-Jimenez for technical assistance. This study was supported by MICIN: RTI2018-099357-B-I00 and HFSP (RGP0016/2018). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovaci&#243;n y Universidades (MCNU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Figure 2 created with BioRender.com.

All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by the institutional ethics committee of the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain, in accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and with the Spanish Royal Decree of 1 February 2013 (53/2013). All efforts were made to minimize the number of animals used and their suffering.
NOTE: This comparative assay to study the segmentation of mitochondrial CoQ pools is described as follows:
1. Protein quantification
<ol>
	<li>Freeze and thaw the isolated mitochondria26 from a wild-type mouse liver three times (i.e., mitochondrial membranes) before experimentation to make the organelles permeable to the reaction substrates.</li>
	<li>Quantify the protein amount of the isolated mitochondria sample by Bradford or Bicinchoninic acid (BCA) methods. In the case of Bradford, add 2 &#181;L of sample into 1 mL of 1x Bradford reagent.</li>
	<li>Split the sample into four subsamples of 20 &#181;g each (namely: A, B, C, D; Figure 2A).</li>
</ol>
2. Measuring CI+CIII activity
NOTE: This part of the protocol uses samples A and B to measure CI+CIII activity (Figure 2B).
<ol>
	<li>Split samples A and B into two subsamples of 10 &#181;g each (namely A1, A2, B1, and B2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 10 &#181;L of 100 mM malonate, and add preheated C1/C2 buffer (Table 1) at 37 &#176;C up to 980 &#181;L (979 &#181;L for cuvettes A2 and B2). 
	CAUTION: This step involves the use of the toxic reagents malonate and potassium cyanide. 
	NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K2HPO4 solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes A1 and A2, and add 10 &#181;L of 1 M NaCl in cuvettes B1 and B2.</li>
	<li>Add 1 &#181;L of 1 mM rotenone into the cuvette containing subsamples A2 and B2. 
	CAUTION: This step involves the use of the toxic reagent rotenone.</li>
	<li>Right before the measurement, add 10 &#181;L of NADH (10 mM) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette by carefully flipping it three times. Place it in the absorbance cuvette reader (UV/VISJASCO spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.</li>
	<li>At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.</li>
</ol>
3.&#160;Measuring CII+CIII activity
NOTE: This part of the protocol uses samples C and D to measure CII+CIII activity (Figure 2C).
<ol>
	<li>Split samples C and D into two subsamples of 10 &#181;g each (namely C1, C2, D1, and D2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 1 &#181;L of 1 mM rotenone, and add preheated C1/C2 buffer at 37 &#176;C up to 980 &#181;L (970 &#181;L for cuvettes C2 and D2). 
	CAUTION: This step involves the use of the toxic reagents potassium cyanide and rotenone. 
	NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K2HPO4 solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes C1 and C2, and add 10 &#181;L of 1 M NaCl in cuvettes D1 and D2.</li>
	<li>Add 1 &#181;L of 1 mM antimycin A into the cuvette containing subsamples C2 and D2. 
	CAUTION: This step involves the use of the toxic reagent antimycin A.</li>
	<li>Right before the measurement, add 10 &#181;L of succinate (1 M) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette carefully, flipping it three times. Place it in the absorbance cuvette reader (UV/VIS spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.</li>
	<li>At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.</li>
</ol>

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The authors declare no conflicts of interest.

Though this protocol represents a very straightforward procedure to identify the existence of the partially segmented CoQ pools, there are a few critical steps to take into account. Substrates (i.e., NADH or succinate) are preferably added last since autooxidation of these compounds may occur. Cuvette&#39;s flipping must be careful in order to avoid the formation of bubbles which may interfere with the reading.
In addition, the present technique presents a few limitations which are worth menti...

Mitochondrial oxidative phosphorylation system (OXPHOS) is the main pathway driving adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) production, and consumption of reducing equivalents, such as nicotinamide adenine dinucleotide (NADH) or succinate, by mitochondria. OXPHOS system is composed of five protein complexes: Complex I (CI) oxidizes NADH and reduces CoQ into ubiquinol (CoQH2). Complex II (CII) oxidizes succinate into fumarate and reduces CoQ into CoQH2. Complex III (CIII) oxidizes CoQH2 back into CoQ, reducing cytochrome c (cyt c). Finally, complex IV (CIV) oxidizes cyt c and reduces oxygen to water. This oxidoreduction chain, the so-called electron transport chain (mETC), is coupled to pumping of H+ across the IMM, which creates an electrochemical gradient used by complex V (CV) to phosphorylate adenosine diphosphate (ADP) into ATP.
mETC complexes can either be alone in the IMM or assemble into quaternary structures called supercomplexes. CIV can assemble with CIII, forming the III2+IV or Q-respirasome (as it is able to respire in the presence of CoQH2)1,2,3 or forming homodimers or homooligomers4. CIII can interact with CI, forming the supercomplex I+III25. Finally, CI is also able to interact with the Q-respirasome, building the I+III2+IV or N-respirasome (as it can respire consuming NADH)1,6,7,8,9,10.
CoQ and cyt c are mobile electron carriers in charge of transferring electrons from CI/CII to CIII, and from CIII to CIV, respectively. Whether or not supercomplexes impose a functional local restriction for these carriers has been a matter of intense debate through the last two decades2,7,11,12,13,14,15,16,17. However, several independent groups have demonstrated that CoQ and cyt c can be functionally segmented into pools in the IMM. In respect of the CoQ, it can be functionally segmented into a specific CoQ pool for CI (CoQNAD) and another pool dedicated to FAD-dependent enzymes (CoQFAD)1,7,12,18,19. However, in order to differentiate the existence of partially segmented functional CoQ pools, the overexpression of the alternative oxidase (AOX) and the generation of specific mtDNA mutants, which can assemble CI in the absence of CIII, were required1,19,20.
The mechanism of reactive oxygen species (ROS) production during hypoxia was unknown until recently. Upon acute hypoxia, CI undergoes the active/deactive (A/D) transition, which involves the decrease in its H+ pumping NADH-CoQ oxidoreductase activity. Such a decrease in H+ pumping acidifies the mitochondrial matrix and partially dissolves the calcium-phosphate precipitates in the mitochondrial matrix, releasing soluble Ca2+. This increase in soluble Ca2+ activates the Na+/Ca2+ exchanger (NCLX), which extrudes Ca2+ in exchange for Na+. Mitochondrial Na+ increase interacts with phospholipids in the inner side of the IMM, decreasing its fluidity and CoQ transfer between CII and CIII, finally producing superoxide anion, a redox signal21. Interestingly, CoQ transfer was only diminished between CII and CIII, but not between CI and CIII, highlighting that 1) Na+ was able to modulate only one of the existing CoQ pools in the mitochondria; 2) there exists functionally differentiated CoQ pools in the IMM. Thus, a widely used protocol for the study of mitochondrial enzyme activities can be used to assess the existence of the mentioned CoQ pools.
The current protocol is based on the measurement of the reduction of oxidized cyt c, the substrate of CIII, by absorbance in the presence of succinate (i.e., CII substrate) or NADH (i.e., CI substrate). The same sample is divided into two, one of which will be treated with KCl, and the other one with the same concentration of NaCl. In this way, given that Na+ decreases IMM fluidity, if CoQ existed in a unique pool in the IMM, both CI+CIII and CII+CIII would decrease in the presence of Na+. However, if CoQ existed in partially segmented functional CoQ pools, the effect of Na+ would mostly (or only) be evident on the CII+CIII activity, but not on the CI+CIII. As recently published21, Na+ only affects the CoQ transfer between CII and CIII (Figure 1C,D), but not between CI and CIII (Figure 1A,B).
This protocol, together with a panoply of techniques, has been used to confirm the existence of partially segmented functional CoQ pools in the IMM, one dedicated to CI (i.e., CoQNAD), and another dedicated to FAD-linked enzymes (i.e., CoQFAD)1,3,7; an observation that, though it continues to be debated22, has been corroborated independently by several groups7,19. Thus, the superassembly of CI into supercomplexes impacts on the local mobility of CoQ, facilitating its usage by the CIII within the supercomplex1,7,13,14,23,24,25.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>10775835001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford protein assay</td>
			<td>Bio-Rad</td>
			<td>5000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c from equine heart</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Malonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1296</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Roche</td>
			<td>10107735001</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium cyanide</td>
			<td>Sigma-Aldrich</td>
			<td>207810</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra Manager software</td>
			<td>JASCO</td>
			<td>version 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>UV/VISJASCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>398055</td>
			<td></td>
		</tr>
	</tbody>
</table>

inner mitochondrial membrane sensitivity to na+ reveals partially segmented functional coq pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Typical results from this protocol are represented below (Figure 3). As reduced cyt c absorbance locates at 550 nm, all uninhibited subsamples must show an increase in the absorbance at 550 nm. Inhibited subsamples ideally show a flat-line or slightly increasing slope (Figure 3). Slopes from inhibited subsamples are to be subtracted from uninhibited subsamples.
Samples A and B, both corrected by their correspondent inhibition and whic...

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Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

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Research

JoVE Journal

Biochemistry

1.8K Views.  Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III CNIC. This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Video: Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...