Protein interactions: analysis using allele libraries.

Interaction defective alleles (IDAs) are alleles that contain mutations affecting their ability to interact with their wild type binding partners. The locations of the mutations may lead to the identification of protein interaction domains and interaction interfaces. IDAs may also distinguish different binding interfaces of multidomain proteins that are part of large complexes, thus shedding light on large protein structures that have yet to be determined. IDAs may also be used in conjunction with RNAi to dissect protein interaction networks. Here, the wild type allele is knocked down and replaced with an IDA that has lost the ability to interact with a specific binding partner. As a result, interactions are disrupted rather than knocking out the entire gene. Thus, IDAs have the potential to be extremely valuable tools in protein interaction network analysis. IDAs can be isolated by reverse two-hybrid analysis, which was demonstrated over a decade ago, but high background levels caused by truncated IDAs have prevented its widespread adoption. We recently described a novel method for full-length allele library generation that eliminates this background and increases the efficiency of the reverse two-hybrid protocol (and IDA isolation) significantly. Here we discuss our strategy for allele library generation, the potential uses of IDAs as outlined above, and additional applications of allele libraries.

A novel approach for generating full-length, high coverage allele libraries for the analysis of protein interactions.

The yeast reverse two-hybrid method was developed to identify mutations disrupting protein-protein interactions. Adoption of the method has been slow, in large part, due to the high frequency of truncation and frameshift mutants typically observed with current protocols. We have developed a new strategy, based on in vitro recombinational cloning and full-length selection in Escherichia coli, to eliminate this background and dramatically increase the efficiency of the reverse two-hybrid protocol. The method was tested by generating an allele library of MyoD1 and selecting for alleles with defective interaction with Id1. Our results confirm that most of the interaction-defective alleles contain a single point mutation in the known interaction domain, the basic helix-loop-helix region. Moreover analysis of the crystal structure of MyoD reveals that the majority of these mutations occurred at the interaction interface. The results obtained using this novel approach for allele library generation demonstrate a significant advancement in the application of yeast reverse two-hybrid screens. Furthermore this method is applicable to any loss-of-function mutant screen where truncated proteins are a source of high background.

C. elegans GLA-3 is a novel component of the MAP kinase MPK-1 signaling pathway required for germ cell survival.

During oocyte development in Caenorhabditis elegans, approximately half of all developing germ cells undergo apoptosis. While this process is evolutionarily conserved from worms to humans, the regulators of germ cell death are still largely unknown. In a genetic screen for novel genes involved in germline apoptosis in Caenorhabditis elegans, we identified and cloned gla-3. Loss of gla-3 function results in increased germline apoptosis and reduced brood size due to defective pachytene exit from meiosis I. gla-3 encodes a TIS11-like zinc-finger-containing protein that is expressed in the germline, from the L4 larval stage to adulthood. Biochemical evidence and genetic epistasis analysis revealed that GLA-3 participates in the MAPK signaling cascade and directly interacts with the C. elegans MAPK MPK-1, an essential meiotic regulator. Our results show that GLA-3 is a new component of the MAPK cascade that controls meiotic progression and apoptosis in the C. elegans germline and functions as a negative regulator of the MAPK signaling pathway during vulval development and in muscle cells.