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

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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.

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Acknowledgments

We owe special thanks to Farhah Assaad for critical reading the first version of the manuscript and Alfons Gierl for generously supporting our projects. We also thank the Deutsche Forschungsgemeinschaft for financial support of this work (DFG To134/2-1 and partially To134/5-2).

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  1. Sabine Schönhofer-Merl

    Present address: Deutsches Herzzentrum München, Klinikum rechts der Isar, Technischen Universität München, Munich, Germany

Authors and Affiliations

  1. Lehrstuhl für Genetik, Wissenschaftszentrum Weihenstephan, Technische Universität München, Emil-Ramann-Str. 8, 85350, Freising, Germany

    Sabine Schönhofer-Merl & Ramón A. Torres-Ruiz

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  1. Sabine Schönhofer-Merl
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  2. Ramón A. Torres-Ruiz
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Correspondence to Ramón A. Torres-Ruiz.

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Communicated by A. Schnittger.

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438_2010_528_MOESM1_ESM.jpg

Predicted trans-membrane domains (Tms) in PEP/PAS2 (“TmConsens”). Shown are predicted Tms in the PEP/PAS2 amino acid sequence (double arrows) and their scores as estimated by the Aramemnon tool. Two weak Tms are indicated by double arrows with stipple lines (top). The consensus scores for the respective predicted Tms (boxes) are given (bottom). For details see text. (JPEG 862 kb)

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Schönhofer-Merl, S., Torres-Ruiz, R.A. The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2. Mol Genet Genomics 283, 439–449 (2010). https://doi.org/10.1007/s00438-010-0528-5

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  • DOI: https://doi.org/10.1007/s00438-010-0528-5

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