Membrane Protein Crystallization

Hunte, Carola; Michel, Hartmut

doi:10.1016/b978-012361776-7/50008-5

Cited by 18 publications

(11 citation statements)

References 64 publications

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“…General and detailed methods for the use of detergents in the purification and crystallization of integral membrane proteins are the subject of an excellent review in this series ( UNIT 4.8 ) and helpful discussions elsewhere (e.g., Ben-Shem et al, 2003; Hunte et al, 2003; Luckey, 2008). The present unit focuses on specific problems involving the use of detergents in structure-function studies of (1) the transmembrane α-helical hetero-oligomeric cyanobacterial cytochrome b 6 f complex (Cyt b 6 f ; Kurisu et al, 2003b; Yamashita et al, 2007; Baniulis et al, 2009), for which general details of preparation and crystallization have been described (Baniulis et al, 2011); and (2) β-barrel proteins from the Escherichia coli outer membrane, which have been structurally characterized in the context of the cellular import mechanism of cytotoxic colicin.…”

Section: Introductionmentioning

confidence: 99%

Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

et al. 2013

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Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical heterooligomeric integral cytochrome b6f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane β-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6f complex, are described. Differences in detergent strategies for isolation and crystallization of β-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented.

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Section: Introductionmentioning

confidence: 99%

Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

et al. 2013

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“…Internal protein sequences were obtained as described previously (32). Briefly complex V subunits were separated by blue SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Identification of Two Proteins Associated with Mammalian ATP Synthase

Meyer

Wittig

Trifilieff

et al. 2007

Molecular & Cellular Proteomics

110

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Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF 1 inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes.

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“…The membranes were centrifuged at 100,000 ϫ g for 1 h, followed by solubilization in 25 mM bis-Tris-HCl, pH 7.0, 2% digitonin, and 500 mM 6-amino-caproic acid. The lysates were cleared at 100,000 ϫ g for 1 h, and then they were separated by Blue Native-PAGE (Hunte et al, 2003). …”

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The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

Mitrović

Ben-Tekaya

Koegler

et al. 2008

MBoC

117

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Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment. INTRODUCTIONThe secretory pathway of higher eukaryotic cells is composed of the three membrane organelles endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and Golgi (Bonifacino and Glick, 2004;Appenzeller-Herzog and Hauri, 2006). Maintenance of these organelles requires a balance of anterograde (secretory) and retrograde vesicular traffic. Anterograde traffic from ER to ERGIC is mediated by coat protein (COP) II vesicles that form at ER exit sites (Aridor et al., 1995;Zeuschner et al., 2006) and fuse with the ERGIC that consists of a few hundred tubulovesicular membrane clusters in vicinity of ER exit sites (AppenzellerHerzog and Hauri, 2006). Transport from ERGIC to Golgi is mediated by pleomorphic vesicles (Ben-Tekaya et al., 2005) that carry COP I (Presley et al., 1997;Scales et al., 1997), although the mechanism of their formation remains unknown. Retrograde traffic mediated by COP I vesicles can occur from ERGIC or Golgi and recycles membrane proteins that possess either dilysine signals, including ERGIC-53 and KDEL-receptor, or diphenylalanine signals, such as members of the 24 protein family. This rapid COP I-dependent recycling is distinct from the slow Golgi-to-ER recycling of Golgi resident proteins that is COP I independent and can be either constitutive or induced (Storrie, 2005).Major constituents of anterograde and retrograde transport vesicles are transmembrane cargo receptors that mediate protein sorting by linking soluble cargo on the luminal side and coat assembly on the cytoplasmic side. To date, only few cargo receptors have been studied in detail. The polytopic transmembrane protein Erv29p is known to cycle between ER and Golgi in yeast and to operate as a cargo receptor (Belden and Barlowe, 2001). Erv29p is required for efficient packaging of the glycosylated ␣-factor pheromone precursor into COP II vesicles departing from the ER. Maturation of carboxypeptidase Y...

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Membrane Protein Crystallization

Cited by 18 publications

References 64 publications

Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

Identification of Two Proteins Associated with Mammalian ATP Synthase

The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

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