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Query: UNIPROT:P06889 (Mol)
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Ubiquinol-cytochrome c reductase is a crucial integral membrane protein in the mitochondrial respiratory cycle. Eleven subunits containing three cytochrome heme groups and a 2Fe-2S Rieske center make up this 240 kDa enzyme complex. Previously, many different crystal forms of the bc1 complex have displayed diffraction to as far as 4.5 A. However, rapid degradation of the protein in the X-ray beam at room temperature has obstructed the collection of a full data set from a single crystal. As slight heterogeneities between crystals severely hampered merging of data from different crystals, we sought a method to stabilize the protein crystal in the X-ray beam in order to collect a full data set from one crystal sample. To this end, water soluble protein crystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In this communication, we report on a successful experiment in which flash-cooled bc1 membrane protein crystals have given rise to sustained diffraction over a 60 hour data collection period at a synchrotron source. Furthermore, we present an improved purification and crystallization protocol yielding crystals readily diffracting out to 3.3 A. These results should greatly aid in the future realization of the molecular structure of the bc1 complex as well as other membrane proteins.
J Mol Biol 1995 Sep 08
PMID:Preliminary cryocrystallographic study of the mitochondrial cytochrome bc1 complex: improved crystallization and flash-cooling of a large membrane protein. 766 27

The subcellular distribution of the two isozymes of 5 alpha-reductase has been controversial. To resolve this issue which could provide clues about the respective functions of the two isozymes, two antisera were generated, one which was specific for the Type 1 5 alpha-reductase and one which recognized both isozymes. In COS cells transfected separately with the Type 1 or Type 2 cDNA, both isozymes were detected on Western blots at an M(r) of 26,000. Subfractionation of the COS cells resulted in the partitioning of both isozymes between the crude nuclear and cytosolic fractions, while cytoimmunofluorescence localized both reductases to the nuclear periphery. In rat liver homogenate, the 5 alpha-reductase was also detected at M(r) 26,000. The 5 alpha-reductase immunoreactivity was increased after castration of the animals with no further effect when castrated animals were treated with androgens. Although the rat liver expresses only the Type 1 5 alpha-reductase, the 5 alpha-reductase was distributed about equally between crude nuclear and cytosolic subfractions; this distribution could be shifted to the cytosolic fractions with harsher homogenization procedures. Further extensive subfractionation and extraction studies identified the rat liver Type 1 5 alpha-reductase as an integral membrane protein present in the outer nuclear membrane of the nuclear envelope and in rough endoplasmic reticulum. Thus, the subfractionation and cytoimmunofluorescence studies are consistent with the localization of the Type 1 5 alpha-reductase to the outer nuclear membrane of the nuclear envelope which is continuous with and indistinguishable from the endoplasmic reticulum. This study is the first to localize rat liver Type 1 5 alpha-reductase to the nuclear envelope to which the prostatic 5 alpha-reductase activity previously had been localized. We conclude that, contrary to previous tissue distribution studies, but consistent with investigations in transfected cells, both isozymes are similarly localized to the nuclear periphery.
Mol Cell Endocrinol 1995 Apr 28
PMID:5 alpha-Reductase type 1 is localized to the outer nuclear membrane. 767 44

The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site. KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using beta-lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV-SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.
Mol Microbiol 1994 Dec
PMID:Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1. 771 49

Recent studies suggest that taurine (2-aminoethanesulfonic acid) is involved in the regulation of protein phosphorylation in excitable tissues such as the retina, brain and heart. In order to determine the structural requirements for the effect of taurine on the phosphorylation of a 44 kDa protein(s), a series of taurine analogues were tested in an in vitro assay using a subcellular mitochondrial fraction of rat heart. Inhibitors of the phosphorylation of the 44 kDa protein include taurine and close structural analogues of taurine such as aminoethylhydrogen sulfate and alpha-sulfo-beta-alanine. Secondary amines with the taurine structure partially locked into a saturated 5-membered ring such as (+/-)piperidine-3-sulfonic acid and 1,2,3,4-tetrahydroquinoline-8-sulfonic acid also possess inhibitory activity. Sulfone analogues of taurine such as 2-aminoethylmethylsulfone, a non-restricted taurine analogue with maximal conformational flexibility about its amino and sulfone moieties, and (+/-)3-aminotetrahydrothiopyran-1,1-dioxide, an analogue containing the sulfone moiety in a six-membered ring structure, were found to be more potent inhibitors of phosphorylation than taurine despite the fact that the sulfone moiety is neither an isosteric nor isoelectronic substitution for the sulfonic acid moiety. The results of this study indicate that the inhibition of the phosphorylation of the 44 kDa protein in a rat heart mitochondrial fraction is relatively specific for the taurine structure. Two analogues of taurine with unsaturated rings containing a primary sulfonic acid and a secondary amine, pyridine-3-sulfonic acid and quinoline-8-sulfonic acid, were observed to be stimulators of the phosphorylation of the 44 kDa protein. In addition, 2-aminobenzenesulfonic acid also stimulated phosphorylation. Phase separation experiments with Triton X-114 suggest that the 44 kDa phosphoprotein is a soluble protein and not an integral membrane protein of the mitochondria. Phosphate incorporation into specific amino acids was determined by two-dimensional electrophoresis on celluloses plates and was found exclusively in the serine residues.
J Mol Cell Cardiol 1994 Dec
PMID:Effects of taurine and taurine analogues on the phosphorylation of a 44 kDa protein present in a mitochondrial subfraction of the rat heart: partial characterization of the 44 kDa phosphoprotein. 773 Oct 61

Nodulin 26 is an integral membrane protein of the symbiosome membrane of nitrogen-fixing soybean nodules. We expressed a nodulin 26 cDNA in transgenic tobacco (TN26 tobacco) under the control of the cauliflower mosaic virus 35S promoter to study subcellular targeting and the physiological effect(s) of its expression. Based on Northern and Western blots, the expression of nodulin 26 mRNA and protein in transgenic plants is high in apical shoot sections, flowers, and stems, low in mature leaves, and absent in roots. Western blot analysis revealed high levels of transgenic nodulin 26 protein in tonoplast membranes. In contrast, nodulin 26 protein was not found in isolated plasma membranes, the soluble fraction, nor in chloroplast and mitochondria-enriched membrane fractions. About 50-60% of the flowers and pods from TN26 tobacco plants abscised prematurely. Seed capsule size and seed fill per capsule from the remainder of surviving flowers were about 50% of that of control plants. Pollen viability was found to be normal, but flowers from TN26 tobacco plants showed shorter anther filaments compared with control plants. Normal seed production and capsule size was restored by manually crossing the stigmas from TN26 plants with isolated pollen from either transgenic or control plants. Thus, the aberrant filament growth could have resulted in the reproductive defects associated with the plants.
Mol Biol Cell 1995 Jan
PMID:Expression of soybean nodulin 26 in transgenic tobacco. Targeting to the vacuolar membrane and effects on floral and seed development. 774 92

The lipid composition of the brush border membrane (BBM) or apical plasma membrane of enterocytes is characterized by a remarkably high glycosphingolipid content (glycosphingolipid: phospholipid:neutral lipid mole ratio of about 1:1:1). A manifestation of the high glycolipid content of the BBM is the lipid fluidity which is low compared to other mammalian plasma membranes and related to it a steep flexibility gradient: hydrocarbon chain segments close to the lipid-water interface have quasi-crystalline packing while hydrocarbon chain segments close to the centre of the lipid bilayer behave like a fluid. An important function of the BBM is the absorption of dietary lipids. The absorption of cholesterol from bile salt micelles has been shown to be protein-mediated. The integral membrane protein responsible for this activity has features similar to non-specific lipid transfer proteins. Another remarkable property of the BBM is described here: phospholipids are exchanged between the lipid bilayer of the BBM and the lipid bilayers of small unilamellar egg phosphatidylcholine (PC) vesicles. In the course of this probably 1:1 exchange, endogenous BBM phospholipids move out of the BBM and the lipid loss is compensated by the insertion of exogenous PC from the small unilamellar vesicles. This exchange activity is probably due to the same protein(s) responsible for lipid absorption in this membrane or at least related to the absorptive capacity of the BBM. The unique feature of small intestinal BBM is that the on- and off-rate of certain lipids is remarkably high: the underlying structure of this activity is still unknown.
Mol Membr Biol
PMID:A unique feature of lipid dynamics in small intestinal brush border membrane. 776 68

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.
Mol Biol Cell 1995 Feb
PMID:Molecular cloning and characterization of human kinectin. 778 43

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.
Mol Biol Cell 1995 Feb
PMID:Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis. 778 44

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 bp upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.
Mol Gen Genet 1994 Nov 01
PMID:PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast. 781 36

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.
Mol Biol Cell 1994 Sep
PMID:Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex. 784 20


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