The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.

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The Elastin-binding Protein ofStaphylococcus aureus(EbpS) Is Expressed at the Cell Surface as an Integral Membrane Protein and Not as a Cell Wall-associated Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m107621200 ◽

2001 ◽

Vol 277 (1) ◽

pp. 243-250 ◽

Cited By ~ 80

Author(s):

Robert Downer ◽

Fiona Roche ◽

Pyong Woo Park ◽

Robert P. Mecham ◽

Timothy J. Foster

Keyword(s):

Cell Wall ◽

Membrane Protein ◽

Cell Surface ◽

Binding Protein ◽

Integral Membrane Protein ◽

Ofstaphylococcus Aureus ◽

A Cell ◽

Elastin Binding Protein

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Engineered Peptide Barcodes for In-Depth Analyses of Binding Protein Ensembles

10.1101/287813 ◽

2018 ◽

Cited By ~ 1

Author(s):

Pascal Egloff ◽

Iwan Zimmermann ◽

Fabian M. Arnold ◽

Cedric A. J. Hutter ◽

Damien Morger ◽

...

Keyword(s):

Mass Spectrometry ◽

Adverse Effects ◽

Membrane Protein ◽

Binding Protein ◽

Integral Membrane Protein ◽

Human Pathogen ◽

Cellular Environment ◽

Protein Ensembles ◽

Living Organisms ◽

Selection Of

ABSTRACTBinding protein generation relies on laborious screening cascades that process candidate molecules individually. To break with this paradigm, we developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without handling individual clones at any stage of the process. NestLink builds on genetically fused barcoding peptides, termed flycodes, which are designed for maximal detectability by mass spectrometry and serve as unique molecular identifiers for accurate deep sequencing. We applied NestLink to overcome current limitations of binder generation. Rare binders against an integral membrane protein were identified directly in the cellular environment of a human pathogen. Hundreds of binder candidates were simultaneously ranked according to kinetic parameters. Adverse effects of target immobilization were overcome by selecting nanobodies against an ABC transporter entirely in solution. NestLink may provide a basis for the selection of tailored binder characteristics directly in tissues or in living organisms.

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Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.2041 ◽

1990 ◽

Vol 111 (5) ◽

pp. 2041-2052 ◽

Cited By ~ 59

Author(s):

L Thomas ◽

H Betz

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Binding Protein ◽

Primary Cultures ◽

Specific Interaction ◽

Integral Membrane Protein ◽

Synaptic Plasma Membrane ◽

Plasma Membrane Protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.

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Detergent Screening for NMR-Based Structural Study of the Integral Membrane Protein, Emopamil Binding Protein (Human Sterol Δ8-Δ7 Isomerase)

Journal of the Korean Magnetic Resonance Society ◽

10.6564/jkmrs.2017.21.1.013 ◽

2017 ◽

Vol 21 (1) ◽

pp. 13-19

Author(s):

Hyung-Sik Won

Keyword(s):

Membrane Protein ◽

Structural Study ◽

Binding Protein ◽

Integral Membrane Protein

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Molecular Characterization of a High-Affinity Xylobiose Transporter of Streptomyces thermoviolaceus OPC-520 and Its Transcriptional Regulation

Journal of Bacteriology ◽

10.1128/jb.186.4.1029-1037.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1029-1037 ◽

Cited By ~ 35

Author(s):

Hiroshi Tsujibo ◽

Mitsuo Kosaka ◽

Sadao Ikenishi ◽

Takaji Sato ◽

Katsushiro Miyamoto ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Gene Cluster ◽

Degradation Products ◽

Binding Protein ◽

Integral Membrane Protein ◽

Upstream Region ◽

Atp Binding ◽

Uptake System ◽

Xylan Degradation

ABSTRACT Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an α-l-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular β-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed high-level affinity for xylobiose (Kd = 8.75 × 10−9 M) and for xylotriose (Kd = 8.42 × 10−8 M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the −10 region of the bxl operon. The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose. This site was also present in the region located between stxI and stxIV and in the upstream region of stxII. BxlR specifically bound to the regions containing the inverted sequence. These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S. thermoviolaceus OPC-520.

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