A guideline and a sample protocol is provided for analysis of protein-protein interactions when at least one protein is a resident of the inner nuclear membrane.
Method Article
A two hybrid test for interaction partners of inner nuclear membrane resident proteins
https://doi.org/10.1038/protex.2016.040
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A guideline and a sample protocol is provided for analysis of protein-protein interactions when at least one protein is a resident of the inner nuclear membrane.
The classical two hybrid system (1) provides an easy means for mapping binary interactions in the global interactomes, screening for new interactors of a protein of interest and testing specific interactions. A major pitfall of this system is the significant probability of false positive and false negative results. These types of errors are especially abundant in the case of integral membrane proteins since their hydrophobic portions tend to demonstrate somewhat nonspecific binding to each other. A common usual way to overcome this problem is to employ protein fragments originated from extramembranous loops. The more complicated split-ubiquitin system was proposed for protein-protein interactions of transmembrane proteins (2). However, these approaches also have their own limitations, leading to large numbers of false positives and false negatives.
Here we provide a guideline and a sample protocol to test for interaction partners of proteins that are natural residents of the inner nuclear membrane and contain at least one nucleoplasmic domain. Most importantly, in the case of these proteins, the full-length polypeptide chain, including intramenbrane segments, can be effectively used as “bait” for the search of protein partners.
In our opinion, this approach may produce more reliable data in comparison with that obtained using protein fragments as baits. We have demonstrated usefulness of the classical yeast two hybrid system for membrane proteins containing nucleoplasmic domains as exemplified by identification of protein partners of the BetaM protein (product of mammalian ATP1B4 gene), which is a natural inner nuclear membrane protein with N-terminus exposed into the intranuclear space (3).
pGBKT7 and pGADT7-RecAB vectors (Clontech)
AH109 yeast strain
YPAD medium
LiAc buffer (0.1 M lithium acetate buffered with 10 mM Tris-HCl at pH 7.5), sterile
PEG-3350 (Sigma)
Denatured salmon sperm DNA (2.0 mg/ml)
Sterile water
SD agar plates -Leu,-Trp plates
SD agar plates -Leu,-Trp plates supplemented with α-X-Gal
SD agar plates -Leu, -His, -Trp
SD agar plates -Ade, -Leu, -His, -Trp supplemented with α-X-Gal
42˚C water bath
30˚C incubator shaker
Clone your protein of interest in the pGADT7-RecAB vector, and two or more interactor proteins into the pGBKT7 vector using standard procedures in E. coli and proceed to yeast transformation.
b) SD -Leu,-Trp plates supplemented with α-X-Gal (plate for growth-independent test for α-galactosidase)
c) SD -Ade, -LEU, -HIS, -TRP supplemented with α-X-Gal (high stringency plates).
6-9 days
The specificity of interaction may be judged from a comparison of two interaction pairs where one serves as a negative control. Typically, both co-transformants should demonstrate good growth on (-Leu,-Trp) plates with blue color developed only in the case of the true-positive interaction. Ideally, there may be a significant difference in growth rate of the clones on the low stringency medium (-LEU, -HIS, -TRP). This is, however, quite rare in the case of membrane proteins, usually unspecific activation of the His gene leads to observable growth. Thus, the most important part is the growth on the high stringency medium (-Ade, -LEU, -HIS, -TRP, +α-X-gal). The test should be considered positive for a pair of proteins if the contransformant grows on all of the aforementioned media and produce blue color in the presence of α-X-gal, whereas the control pair shows no observable growths on the high stringency medium and no blue pigment.
Supported by the Russian Foundation for Basic Research (grant 14-04-01844), and by the Department of Physiology and Pharmacology and Center for Diabetes and Endocrine Research, University of Toledo College of Medicine.
The authors declare no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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