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Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange

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Abstract

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrPC), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP91. Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.

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Abbreviations

TM:

Transmembrane

ER:

Endoplasmic reticulum

PM:

Plasma membrane

FP:

Fluorescent protein

GFP:

Green fluorescent protein

YFP:

Yellow fluorescent protein

GPCR:

G protein-coupled receptor

sst:

Somatostatin receptor

PrP:

Prion protein

GPBP:

Goodpasture antigen-binding protein

HBSS:

Hanks’ balanced salt solution

PEA:

pH exchange assay

pHo :

Extracellular pH

pHi :

Intracellular pH

ROI:

Region of interest

GPI:

Glycosylphosphatidylinositol

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Acknowledgments

The authors thank Dr. Juan Saus for the gift of reagents and helpful discussions on GPBP biochemistry and C. Mark for editorial assistance. BD was supported by FISCAM (MOV-2006_IE/07), MD-P by MICINN (Sara Borrell Program CD07/00246) and TF by MICINN (Ramón y Cajal contract). This work was supported by grants from the Spanish Ministry of Science and Innovation MICINN/FEDER BFU2008-03288 (JL), SAF2006-00418 and BFU2009-07971 (MG), BFU2007-60180 (JPC), the Junta de Andalucía BIO-0139 and CTS-01705 (JPC), European Union MRTN-2005-019481 (JL) and MIRG-CT-2006-026702 (TF) and Instituto de Salud Carlos-III FIS-PI052270 (TF).

Conflict of interest statement

MD-P and JPC are listed as the inventors of patent PCT/ES2007/00627 for the commercial use of sst5TMD4. The remaining co-authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Centro Regional de Investigaciones Biomédicas y Facultad de Medicina, Universidad de Castilla-La Mancha, 02006, Albacete, Spain

    Beatriz Domingo & Juan Llopis

  2. Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006, Madrid, Spain

    María Gasset

  3. Instituto de Parasitología y Biomedicina López-Neyra, CSIC, 18100, Granada, Spain

    Mario Durán-Prado

  4. Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Edificio Severo Ochoa, Planta 3. Campus de Rabanales, Córdoba, Spain

    Justo P. Castaño

  5. Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain

    Justo P. Castaño

  6. CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn 06/03), Córdoba, Spain

    Justo P. Castaño

  7. Hospital 12 de Octubre, 28041, Madrid, Spain

    Antonio Serrano

  8. Centro Nacional de Biotecnología, CSIC, 28049, Madrid, Spain

    Thierry Fischer

Authors
  1. Beatriz Domingo
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  2. María Gasset
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  3. Mario Durán-Prado
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  4. Justo P. Castaño
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  5. Antonio Serrano
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  7. Juan Llopis
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Corresponding author

Correspondence to Juan Llopis.

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Supplementary material 1 (DOC 26 kb)

Fig. S1

. The changes imposed on pHi by perfusing HeLa cells with null buffers were calibrated with the ratiometric pH indicator BCECF using nigericin/monensin. (A) BCECF excitation ratio (500 nm/430 nm). (B) Calibrated pH of the same experiment. (TIFF 70 kb)

Fig. S2

. Determination of PrP-YFP topology on the PM of Be2C cells by pHo exchange. *p = 0.0392. (TIFF 84 kb)

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Domingo, B., Gasset, M., Durán-Prado, M. et al. Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange. Cell. Mol. Life Sci. 67, 3345–3354 (2010). https://doi.org/10.1007/s00018-010-0386-7

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  • DOI: https://doi.org/10.1007/s00018-010-0386-7

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