Artificial Neural Network for the Prediction of Tyrosine-Based Sorting Signal Recognition by Adaptor Complexes
Figure 1
Two-hybrid approach and result coding. (a) Clathrin-associated adaptor complexes bind Y-signals. Scheme depicts a Y-signal (fitting into a XXXYXXØ consensus) within the cytoplasmic tail of a transmembrane protein bound by an adaptor complex (AP in orange). X represents any aminoacid and Ø a residue with a bulky hydrophobic side chain (F, M, I, L and V). The AP’s -subunits bind signals located at about 6–10 aminoacids from the transmembrane domain. (b) Two-hybrid strategy used in this study. Yeast two-hybrid strain (AH109) bearing-integrated reporter genes were transformed with plasmids expressing the Gal4 binding domain (BD) fused to a XXXYXXØ-signal (via a TGN38-derived spacer) and the Gal4 activation domain (AD) fused to the C-terminus of an AP subunit. The GAL4 upstream activating sequences (UAS) within the reporter gene are bound by the Gal4BD-Y signal fusion. If the expressed subunit binds the featured signal, then the Gal4AD activates the HIS3 open reading frame. His3 production allows the cells to grow in absence of the aminoacid histidine (−His), leading to the formation of colonies. (c) Result coding: The colony formation two-hybrid readout was coded as follows: growth in −His (/Y-signal interaction) = 1, whereas absence of growth in −His (lack of interaction) = 0. The SFYYEEI signal used as example was isolated in a combinatorial two-hybrid screen. Signal’s critical Y and Ø (I in this signal) are indicated in blue and were alternatively mutated to A. The interacting pair mouse p53 and SV40 T-large antigen (TL-Ag) was used as a positive control and as negative control when cotransformed with any other construct.