Isolation Of Membrane Protein Complexes Research Articles

Large-scale growth of C. elegans and isolation of membrane protein complexes.

Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.

Impact of Detergents on Membrane Protein Complex Isolation.

Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that β-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij-35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

Blue Native PAGE: Applications to Study Peroxisome Biogenesis.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Two-dimensional Blue Native/SDS-PAGE Analysis Reveals Heat Shock Protein Chaperone Machinery Involved in Hepatitis B Virus Production in HepG2.2.15 Cells

Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.

Deficiency of the α Subunit of Succinate–Coenzyme A Ligase Causes Fatal Infantile Lactic Acidosis with Mitochondrial DNA Depletion

Fatal infantile lactic acidosis is a severe metabolic disorder characterized by the onset of lactic acidosis within the 1st d of life and early death. We found a combined respiratory-chain enzyme deficiency associated with mitochondrial DNA (mtDNA) depletion in a small consanguineous family with this disorder. To identify the disease-causing gene, we performed single-nucleotide polymorphism homozygosity mapping and found homozygous regions on four chromosomes. DNA sequencing revealed a homozygous 2-bp deletion in SUCLG1, a gene that encodes the alpha subunit of the Krebs-cycle enzyme succinate-coenzyme A ligase (SUCL). The mtDNA depletion is likely explained by decreased mitochondrial nucleoside diphosphate kinase (NDPK) activity resulting from the inability of NDPK to form a complex with SUCL.

High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Two-dimensional Blue Native/SDS Gel Electrophoresis of Multiprotein Complexes from Helicobacter pylori

The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

Analysis of Molecular Masses and Oligomeric States of Protein Complexes by Blue Native Electrophoresis and Isolation of Membrane Protein Complexes by Two-Dimensional Native Electrophoresis

Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form

A discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge shift on the proteins and aminocaproic acid served to improve solubilization of membrane proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts, yeast, and bacteria were applied to the gels. From cells containing mitochondria, all the multiprotein complexes of the oxidative phosphorylation system were separated within one gel. The complexes were resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of all respiratory chain complexes was almost quantitative. The percentage recovery of functional activity depended on the respective protein complex studied and was zero for some complexes, but almost quantitative for others. The system is especially useful for small scale purposes, e.g., separation of radioactively labeled membrane proteins, N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic studies of inborn neuromuscular diseases.