Research Article

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 𝜇 -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 𝜇 subunit. The GAL4 upstream activating sequences (UAS) within the reporter gene are bound by the Gal4BD-Y signal fusion. If the expressed 𝜇 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 ( 𝜇 /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.
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