Improved Split-Ubiquitin Screening Technique to Identify Surface Membrane Protein-Protein Interactions

Abstract

Yeast-based methods are still the workhorse for the detection of protein-protein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the split-ubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae. Here we demonstrate that a modification of the widely used membrane SUS involving the transmembrane (TM) domain of the yeast receptor Wsc1 increases the stringency of screening and improves the selectivity for proteins localized in the plasma membrane (PM).

The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

“…Yeast-based split ubiquitin screen and pair-wise interaction analysis identified CD63 as an interaction partner of gp41 29 , and this finding was confirmed by the correlative FRET-PLA technique 40 . Our previously performed PPI analysis was appropriate to address the question of whether CD63 and gp41 can interact, but it did not provide details on where or under which circumstances the identified interaction happens.…”

“…Using a membrane-based split ubiquitin screen, we have identified full-length CD63 from a Jurkat T cell cDNA library as a binding partner of gp41 29 . This yeast-based screening method is suitable to detect novel interactions between membrane proteins in their native conformation in vivo 30 , 31 .…”

“…1 A) with gp41 were performed. As bait for these experiments, a previously described vector named pBT3-SUC-gp41 was used 29 . It consists of a cDNA fragment encoding the ectodomain and transmembrane region of gp41 (HIV-1 Env aa 534–709) cloned into the pBT3-SUC vector upstream in-frame with the C-terminal part of yeast ubiquitin (C ub ) and the LexA-VP16 reporter cassette.…”

“…Co-expression of the bait along with the control prey Alg5-N ub I resulted in a reconstitution of split-ubiquitin through the strong affinity of wild-type N-terminal part of yeast ubiquitin (N ub I) for C ub and the concurrent activation of reporter genes. In contrast, when the bait is co-expressed together with the negative control prey pDL2-Alg5 reporter genes were not activated because the mutated N ub G (I13G) moiety had no affinity for C ub 29 , 30 , 32 . After generating a different set of CD63 mutant prey vectors, experiments were performed to analyze the interaction of CD63 variants with the bait pBT3-SUC-gp41 in order to identify the CD63 interacting domain.…”

“…For the construction of prey plasmids containing a set of different CD63 domains (the amino acid [AA] sequence length is given in the Supplementary Table 1 ), we used the vectors pPR3-N and pPR3-SUC (Dualsystems Biotech AG, Schlieren, Switzerland). The indicated primers (Supplementary Table 2 ) with variable Sfi l restriction sites 29 at the 5′ and 3′ regions for directional cloning were used to amplify CD63 sequence from the template vector pPR3-N-CD63 29 by using 1 unit of PfuUltra HF polymerase (Agilent Technologies, Böblingen, Germany). The amplicons were digested with the Sfi I restriction enzyme (New England Biolabs, Frankfurt, Germany) overnight at 50 °C.…”