scholarly journals Directed Evolution of a Probe Ligase with Activity in the Secretory Pathway and Application to Imaging Intercellular Protein–Protein Interactions

Biochemistry ◽  
2013 ◽  
Vol 52 (21) ◽  
pp. 3728-3739 ◽  
Author(s):  
Katharine A. White ◽  
Phillip M. Zegelbone
Keyword(s):  
Directed Evolution ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Protein Interactions

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors

2022 ◽  
Author(s):  
Jorge Ripoll-Rozada ◽  
Joshua W. C. Maxwell ◽  
Richard J. Payne ◽  
Pedro José Barbosa Pereira
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Serine Proteinase ◽  
Blood Feeding ◽  
Thrombin Inhibitor ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Human Fibrinogen ◽  
Clotting Cascade ◽  
Bona Fide

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein–protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

scholarly journals Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

PLoS ONE ◽  
2014 ◽  
Vol 9 (12) ◽  
pp. e116234 ◽  
Author(s):  
Kathrin Roderer ◽  
Martin Neuenschwander ◽  
Giosiana Codoni ◽  
Severin Sasso ◽  
Marianne Gamper ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Interactions ◽  
Functional Mapping ◽  
Enzyme Complex ◽  
Protein Protein Interactions

Visualization of protein interactions inside the secretory pathway

2007 ◽  
Vol 35 (5) ◽  
pp. 970-973 ◽  
Author(s):  
B. Nyfeler ◽  
H.-P. Hauri
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Coagulation Factor ◽  
Cdna Libraries ◽  
Major Protein ◽  
Protein Protein Interactions ◽  
A Genome

The ER (endoplasmic reticulum) is a major protein folding and modification organelle. In its lumen, the ER processes a third of all newly synthesized proteins. To accomplish this task, numerous resident proteins capture the nascent and newly synthesized proteins. The underlying luminal protein–protein interactions, however, are inherently difficult to analyse, mainly due to their transient nature and the rather specialized environment of the ER. To overcome these limitations, we developed a PCA (protein fragment complementation assay) based on the citrine variant of YFP (yellow fluorescent protein). YFP PCA was successfully applied to visualize the protein interactions of the cargo transport receptor ERGIC-53 (endoplasmic reticulum–Golgi intermediate compartment protein of 53 kDa) with its luminal interaction partner MCFD2 (multiple coagulation factor deficiency protein 2) and its cargo proteins cathepsin Z and cathepsin C in a specific manner. With the prospect of screening cDNA libraries for novel protein–protein interactions, YFP PCA is a promising emerging technique for mapping protein interactions inside the secretory pathway in a genome-wide setting.

Dissecting Protein−Protein Interactions Using Directed Evolution

Biochemistry ◽  
2011 ◽  
Vol 50 (13) ◽  
pp. 2394-2402 ◽  
Author(s):  
Daniel A. Bonsor ◽  
Eric J. Sundberg
Keyword(s):  
Directed Evolution ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals Assembly of the Yeast Vacuolar H+-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex

1998 ◽  
Vol 142 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Laurie A. Graham ◽  
Kathryn J. Hill ◽  
Tom H. Stevens
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cross Linking ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Atpase Subunit ◽  
Mutant Cells

Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of ∼5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein–protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.

The Organizing Potential of Sphingolipids in Intracellular Membrane Transport

2001 ◽  
Vol 81 (4) ◽  
pp. 1689-1723 ◽  
Author(s):  
Joost C. M. Holthuis ◽  
Thomas Pomorski ◽  
René J. Raggers ◽  
Hein Sprong ◽  
Gerrit Van Meer
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Lipid Phase ◽  
Protein Protein Interactions ◽  
Membrane Changes ◽  
Intracellular Membrane Transport ◽  
Sorting Station ◽  
Lipid Phase Separation ◽  
Surrounding Membrane ◽  
Endocytic Pathways

Eukaryotes are characterized by endomembranes that are connected by vesicular transport along secretory and endocytic pathways. The compositional differences between the various cellular membranes are maintained by sorting events, and it has long been believed that sorting is based solely on protein-protein interactions. However, the central sorting station along the secretory pathway is the Golgi apparatus, and this is the site of synthesis of the sphingolipids. Sphingolipids are essential for eukaryotic life, and this review ascribes the sorting power of the Golgi to its capability to act as a distillation apparatus for sphingolipids and cholesterol. As Golgi cisternae mature, ongoing sphingolipid synthesis attracts endoplasmic reticulum-derived cholesterol and drives a fluid-fluid lipid phase separation that segregates sphingolipids and sterols from unsaturated glycerolipids into lateral domains. While sphingolipid domains move forward, unsaturated glycerolipids are retrieved by recycling vesicles budding from the sphingolipid-poor environment. We hypothesize that by this mechanism, the composition of the sphingolipid domains, and the surrounding membrane changes along the cis- trans axis. At the same time the membrane thickens. These features are recognized by a number of membrane proteins that as a consequence of partitioning between domain and environment follow the domains but can enter recycling vesicles at any stage of the pathway. The interplay between protein- and lipid-mediated sorting is discussed.

Sign in / Sign up

Export Citation Format

Share Document