Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

Crichton, Paul G.; Harding, M.; Ruprecht, Jonathan J.; Lee, Yang; Kunji, Edmund R. S.

doi:10.1074/jbc.m113.484329

Cited by 60 publications

(75 citation statements)

References 49 publications

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“…4A). Minor amounts of protein at higher molecular masses (see gel profile of the eluted fractions) were consistent with various oligomers associated with generic aggregation in detergent, as observed for the ADP/ATP carrier (35,36). Quantification of the associated detergent and lipid present in the UCP1 peak fraction revealed that 2.3 g of detergent and 0.5 g of lipid per g of protein were bound.…”

Section: Resultssupporting

confidence: 70%

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Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Lee

Willers

Kunji

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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122

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Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein.Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.

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Section: Resultssupporting

confidence: 70%

“…In recent years, several studies have demonstrated that the mitochondrial ADP/ATP carrier is monomeric, not dimeric (32)(33)(34)(35)(36), which raises questions on the oligomeric state of UCP1. Resolving the basic functional unit of UCP1 is paramount for understanding its mechanism.…”

Section: Significancementioning

confidence: 99%

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Lee

Willers

Kunji

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

122

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“…20,21 Although the zwitterionic detergent CHAPS is thought to maintain Bax in its inactive state, 5,12 it has also been reported to disrupt Bax interactions, 19 and fails to efficiently solubilize certain MOM proteins such as VDAC1. 22 When solubilized in digitonin, but not CHAPS or Triton X-100, distinct Bax complexes in subcellular fractions (cytosol and mitochondria-enriched heavy membrane) of non-apoptotic or apoptotic cells were observed (Figure 1b). Endogenous Bax in Bak À / À MEFs formed similar complexes to ectopically expressed hBax ( Figure 1c) and none were detected in Bak À / À Bax À / À MEFs ( Figure 1d) thereby confirming specificity.…”

Section: Resultsmentioning

confidence: 97%

“…Increasing digitonin concentrations did not disrupt the high molecular weight form of Bax, but instead slightly retarded the migration of monomeric Bax on BN-PAGE (Figure 1f and Supplementary Figure 3, compare lanes 3 and 7), consistent with a previous report for other membrane-associated proteins. 22 The localization of Bax to a high molecular weight complex in mitochondria of non-apoptotic MEFs was reminiscent of Bak complexing with VDAC2. 20,21 To assess whether Bax also associates with VDAC2, we analysed Vdac2 À / À MEFs.…”

Section: Resultsmentioning

confidence: 99%

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

et al. 2014

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In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x L . Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.

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“…The major and minor bands were ϳ224 kDa and ϳ103 kDa and ϳ248 kDa and ϳ115 kDa in SMS1-and SMS2-expressing cells, respectively. As the migration of membrane proteins is affected by several factors, including lipids, detergents, and Coomassie Blue G250 in BN-PAGE, calibration is crucial in mass estimation (15,16). We used aquaporin-1, which is a well characterized membrane protein that forms tetramers (17), with a known molecular mass to calculate a correction factor.…”

Section: Resultsmentioning

confidence: 99%

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum

Hayashi

Nemoto-Sasaki

Matsumoto

et al. 2017

Journal of Biological Chemistry

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Edited by Dennis R. VoelkerSphingomyelin synthase (SMS) is the key enzyme for crosstalk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the N and C termini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N-and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-⌬C22 and SMS2-⌬C30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.

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Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

Cited by 60 publications

References 49 publications

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum

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