Green fluorescent protein as a noninvasive intracellular pH indicator.

It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.

[1]  R Y Tsien,et al.  Understanding, improving and using green fluorescent proteins. , 1995, Trends in biochemical sciences.

[2]  O. Seksek,et al.  Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry. , 1996, Journal of cell science.

[3]  William W. Ward,et al.  SPECTRAL PERTURBATIONS OF THE AEQUOREA GREEN‐FLUORESCENT PROTEIN , 1982 .

[4]  S. Munro,et al.  A C-terminal signal prevents secretion of luminal ER proteins , 1987, Cell.

[5]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[6]  W. Hauswirth,et al.  A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells , 1996, Journal of virology.

[7]  R Marsault,et al.  Targeting aequorin and green fluorescent protein to intracellular organelles. , 1996, Gene.

[8]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[9]  Y. Kimata,et al.  A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures. , 1997, Biochemical and biophysical research communications.

[10]  G. Nolan,et al.  Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[11]  F. Rottman,et al.  Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism. , 1982, The Journal of biological chemistry.

[12]  J. Rine,et al.  In vivo examination of membrane protein localization and degradation with green fluorescent protein. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[13]  S. Grinstein,et al.  Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor , 1996, The Journal of cell biology.

[14]  S. Boxer,et al.  Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Alexander Wlodawer,et al.  The structural basis for spectral variations in green fluorescent protein , 1997, Nature Structural Biology.

[16]  J. Lippincott-Schwartz,et al.  Diffusional Mobility of Golgi Proteins in Membranes of Living Cells , 1996, Science.

[17]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[18]  C Kaether,et al.  Green fluorescent protein: applications in cell biology , 1996, FEBS letters.

[19]  Roger Y. Tsien,et al.  Crystal Structure of the Aequorea victoria Green Fluorescent Protein , 1996, Science.

[20]  T. Hughes,et al.  The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study , 1997, The Journal of cell biology.

[21]  W. Ward,et al.  Renaturation of Aequorea green-fluorescent protein , 1981 .

[22]  M. Oka,et al.  Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. , 1995, Journal of biochemistry.

[23]  G. Banting,et al.  TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38. , 1996, Journal of cell science.

[24]  Tullio Pozzan,et al.  Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells , 1995, Current Biology.

[25]  G. Phillips,et al.  The molecular structure of green fluorescent protein , 1996, Nature Biotechnology.

[26]  M. Fukuda,et al.  Identification of the full-length coding sequence for human galactosyltransferase (β-N-acetylglucosaminide: β1,4-galactosyltransferase) , 1988 .

[27]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[28]  R Y Tsien,et al.  Wavelength mutations and posttranslational autoxidation of green fluorescent protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Verkman,et al.  Direct Measurement of trans-Golgi pH in Living Cells and Regulation by Second Messengers (*) , 1995, The Journal of Biological Chemistry.

[30]  J A Hammer,et al.  Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein. , 1996, Developmental biology.

[31]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[32]  Roger Y. Tsien,et al.  Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo , 1996, Current Biology.

[33]  Bence Ölveczky,et al.  Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix , 1998, The Journal of cell biology.

[34]  A. Persechini,et al.  Detection in Living Cells of Ca2+-dependent Changes in the Fluorescence Emission of an Indicator Composed of Two Green Fluorescent Protein Variants Linked by a Calmodulin-binding Sequence , 1997, The Journal of Biological Chemistry.

[35]  S J Remington,et al.  Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[36]  A. Verkman,et al.  Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. , 1997, Biophysical journal.

[37]  William W. Ward,et al.  SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN‐FLUORESCENT PROTEINS , 1980 .