Figure 1.

Schematic diagram describing the reverse RRS approach. The membrane protein of interest is expressed in a Cdc25-2 yeast strain with no additional sequences. The bait is located at the membrane thus preserving its functional conformation. (A) The bait protein is not expected to induce cell growth of Cdc25-2 yeast cells at the restrictive temperature. (B) A known protein partner for the bait or a cDNA library is expressed as a membrane fusion with the cytoplasmic oncogenic Ras protein. Unless the cDNA encodes for a membrane protein or is associated with a membrane component it is not expected to result in Ras translocation to the membrane and cell growth at the restrictive temperature. (C) Upon protein–protein interaction between the membrane bait and the protein prey fused to Ras, Ras is translocated to the plasma membrane which allows cell growth at the restrictive temperature.