Yeast two-hybrid, a powerful tool for systems biology - PubMed (original) (raw)

Yeast two-hybrid systems, their subcellular location within a yeast cell, and their operating mode (represented at the moment of bait-prey interaction). Protein X (dark blue puzzle piece, part of bait construct) and protein Y (light blue puzzle piece, part of prey construct) directly interact (fitting puzzle pieces), thus inducing reconstitution of split-proteins (puzzle pieces of different colors in A, D, E), membrane recruitment (B, C) or protein dimerization (F). Protein fusions in bait or prey constructs are shown as solid black lines between puzzle pieces. Bait-prey interaction activates further downstream events (arrows) that directly (A) or indirectly (B, C, D, F) lead to transcriptional activation, or are independent of transcriptional activation (D, E), finally yielding screenable readouts like growth on specific media or color reactions. (A) Nuclear Y2H systems all require protein recruitment and bait-prey interaction at nuclear DNA. The

classic Y2H

and

RTA Y2H

both engage RNA polymerase II (RNA Pol II) transcription either by its activation or its inhibition. By contrast, the

Pol III Y2H

, involves RNA polymerase III (RNA Pol III) transcription. (B) Ras signalling based Y2H at the plasma membrane. The

SRS Y2H

,

RRS Y2H

, and

rRRS Y2H

are all based on protein recruitment to the plasma membrane via bait-prey interaction and subsequent activation of MAPK downstream signalling. While in the SRS and RRS Y2H the prey constructs harboring protein Y are anchored at the membrane via myristoylation to analyze interactions with cytosolic bait constructs harboring protein X, the rRRS is used to analyze interactions between soluble preys containing protein Y and partner X being a membrane protein. (C) G-protein signalling-based Y2H at the plasma membrane. In the

G-protein fusion Y2H

, bait X is a membrane or membrane-associated protein whose interaction with the prey construct disrupts protein G downstream signalling. (D) Split-ubiquitin based Y2H systems involve reconstitution of ubiquitin from two domains upon bait-prey interaction. Their subcellular localization depends on the nature of interacting proteins X or Y, and on the reporter proteins used. The

Split ubiquitin Y2H

uses non-transcriptional reporting of protein interactions in the cytosol, but can also be used for membrane proteins (not shown). The

MbY2H

is used for interaction analysis with membrane baits and thus occurs at the membrane location of protein X, e.g. the plasma membrane. The

CytoY2H

is used for membrane anchored cytosolic baits and occurs close to the ER membrane (E) Split-protein sensor Y2H. The

Split-Trp Y2H

is used to assay cytosolic bait-prey interactions based on reconstitution of an enzyme in tryptophan synthesis, allowing for non-transcriptional reporting. (F) ER Y2H system. The

SCINEX-P Y2H

allows bait-prey interaction analysis in the reducing environment of the ER, based on protein dimerization in unfolded protein signalling. ER, endoplasmic reticulum; for further abbreviations and details see chapter 3.2.