Protein–protein interactions

2010 ◽  
Vol 38 (4) ◽  
pp. 875-878 ◽  
Author(s):  
Mike P. Williamson ◽  
Michael J. Sutcliffe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
High Throughput ◽  
Protein Interactions ◽  
Metabolic Pathway ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Good Evidence ◽  
Protein Protein Interactions ◽  
Two Hybrid

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

scholarly journals A High-ThroughputCandida albicansTwo-Hybrid System

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Floris Schoeters ◽  
Carol A. Munro ◽  
Christophe d’Enfert ◽  
Patrick Van Dijck
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Throughput ◽  
Protein Interactions ◽  
Fungal Pathogen ◽  
Model Organism ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Content Type ◽  
Two Hybrid

ABSTRACTCandida albicansis a human fungal pathogen that does not follow the universal codon usage, as it translates the CUG codon into serine rather than leucine. This makes it difficult to study protein-protein interactions using the standard yeast two-hybrid (Y2H) system in the model organismSaccharomyces cerevisiae. Due to the lack of adapted tools, only a small number of protein-protein interactions (PPIs) have been detected or studied usingC. albicans-optimized tools despite the importance of PPIs to understand cell biology. However, with the sequencing of the whole genome ofC. albicans, the availability of an ORFeome collection containing 5,099 open reading frames (ORFs) in Gateway-adapted donor vectors, and the creation of a Gateway-compatibleC. albicans-specific two-hybrid (C2H) system, it became possible to study protein-protein interactions on a larger scale usingC. albicansitself as the model organism. Erroneous translations are hereby eliminated compared to using theS. cerevisiaeY2H system. Here, we describe the technical adaptations and the first application of the C2H system for a high-throughput screen, thus making it possible to screen thousands of PPIs at once inC. albicansitself. This first, small-scale high-throughput screen, using Pho85 as a bait protein against 1,646 random prey proteins, yielded one interacting partner (Pcl5). The interaction found with the high-throughput setup was further confirmed with a low-throughput C2H experiment and with a coimmunoprecipitation (co-IP) experiment.IMPORTANCECandida albicansis a major fungal pathogen, and due to the rise of fungal infections and emerging resistance to the limited antifungals available, it is important to develop novel and more specific antifungals. Protein-protein interactions (PPIs) can be applied as very specific drug targets. However, because of the aberrant codon usage ofC. albicans, the traditional yeast two-hybrid system inSaccharomyces cerevisiaeis difficult to use, and only a limited number of PPIs have been described inC. albicans. To overcome this, aC. albicanstwo-hybrid (C2H) system was developed in 2010. The current work describes, for the first time, the application of the C2H system in a high-throughput setup. We hereby show the usefulness of the C2H system to investigate and detect PPIs inC. albicans, making it possible to further elucidate protein networks inC. albicans, which has the potential to lead to the development of novel antifungals which specifically disrupt PPIs important for virulence.

scholarly journals A critical and Integrated View of the Yeast Interactome

2004 ◽  
Vol 5 (5) ◽  
pp. 382-402 ◽  
Author(s):  
Michael Cornell ◽  
Norman W. Paton ◽  
Stephen G. Oliver
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
False Positive ◽  
Affinity Purification ◽  
Information Management System ◽  
Positive Interactions ◽  
Global Studies ◽  
Protein Protein Interactions ◽  
Integrative Analyses ◽  
Genome Information

Global studies of protein–protein interactions are crucial to both elucidating gene function and producing an integrated view of the workings of living cells. High-throughput studies of the yeast interactome have been performed using both genetic and biochemical screens. Despite their size, the overlap between these experimental datasets is very limited. This could be due to each approach sampling only a small fraction of the total interactome. Alternatively, a large proportion of the data from these screens may represent false-positive interactions. We have used the Genome Information Management System (GIMS) to integrate interactome datasets with transcriptome and protein annotation data and have found significant evidence that the proportion of false-positive results is high. Not all high-throughput datasets are similarly contaminated, and the tandem affinity purification (TAP) approach appears to yield a high proportion of reliable interactions for which corroborating evidence is available. From our integrative analyses, we have generated a set of verified interactome data for yeast.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

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