The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2

Abstract

We have explored a modified cytosolic yeast-two-hybrid Sos-recruitment system (SRS) in order to test for membrane localization of a protein. In this system, membrane localization is assessed by rescue of a yeast strain carrying a temperature-sensitive mutation in the CDC25 gene (cdc25-2) at restrictive temperature. The homologous human Sos (hSos) is capable to replace cdc25-2 provided that it is attached to the membrane because only then hSos is functional. This can be achieved when hSos is artificially fused to a protein containing trans-membrane domains (Tms). GFP/YFP fusion construct analyses of the Arabidopsis thaliana PEPINO/PASTICCINO2 (PEP/PAS2) protein have previously shown disparate cellular localizations although this protein possesses clear Tms. Analysis of N-terminal and C-terminal hSos-PEP/PAS2 fusions respectively suggests, that PEP/PAS2 is an integral membrane protein with cytosolic N- and C-termini. This implies that the protein has an even number of Tms and that the first Tm, a signal peptide, is not cleaved off. Our study shows that SRS is suitable to test for protein membrane localization and possibly for more detailed topological analysis of membrane proteins.

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

“…5A) provides an interesting assay for interactions when proteins are unable to enter the nucleus or when posttranslational modifications in the cytoplasmic milieu are required. In addition, the method has been applied for the discovery of inhibitors of HIV-1 Gag dimerization (652), the analysis of protein membrane localization, and the determination of numbers of transmembrane domains (297,565). An improved version was created by using mammalian activated Ras (mRas) instead of Sos in the cdc25-2 strain (64).…”