Construction of a directory of tobacco plasma membrane proteins by combined two‐dimensional gel electrophoresis and protein sequencing

The polypeptide pattern of the plasma membrane from tobacco was studied by two‐dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate – polyacrylamide gel electrophoresis (IEF/SDS—PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4–8 immobilized pH gradient (IPG)‐IEF and 10%T SDS‐PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross‐section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in‐gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG‐IEF, and N‐terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG‐IEF gels and in‐gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant‐specific genes.

[1]  M. Rossignol,et al.  Auxin-induced differential sensitivity of the H+-ATPase in plasma membrane subfractions from tobacco cells. , 1996, Biochemical and biophysical research communications.

[2]  M. Rossignol,et al.  Basic plasticity of protein expression in tobacco leaf plasma membrane , 1995 .

[3]  R. Dixon,et al.  Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  U. Fischer,et al.  Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system. , 1994, The Plant journal : for cell and molecular biology.

[5]  J. Jauniaux,et al.  Identification of a high affinity NH4+ transporter from plants. , 1994, The EMBO journal.

[6]  W. Frommer,et al.  Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. I. D. Michelis,et al.  Characteristics and Regulatory Properties of the H+‐ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana , 1993 .

[8]  J. Huet,et al.  Characterization of a Novel Protein Induced by Progressive or Rapid Drought and Salinity in Brassica napus Leaves. , 1992, Plant physiology.

[9]  F. Gaymard,et al.  Cloning and expression in yeast of a plant potassium ion transport system. , 1992, Science.

[10]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[11]  E. Bray.  Drought‐stress‐induced polypeptide accumulation in tomato leaves , 1990 .

[12]  J. Vandekerckhove,et al.  Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[13]  F. Dupont,et al.  The effects of salt stress on polypeptides in membrane fractions from barley roots. , 1988, Plant physiology.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  A. Tsugita,et al.  Separation and characterization of Arabidopsis thaliana proteins by two‐dimensional gel electrophoresis , 1995, Electrophoresis.

[16]  M. Sussman.  Molecular Analysis of Proteins in the Plant Plasma Membrane , 1994 .

[17]  Thierry Rabilloud,et al.  Sample application by in‐gel rehydration improves the resolution of two‐dimensional electrophoresis with immobilized pH gradients in the first dimension , 1994, Electrophoresis.

[18]  M J Dunn,et al.  Positional reproducibility of protein spots in two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: An interlaboratory comparison , 1994, Electrophoresis.

[19]  M. Sussman.  Molecular analysis of proteins in the plasma membrane , 1994 .

[20]  D. Hochstrasser,et al.  A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale , 1993, Electrophoresis.

[21]  M. Filali,et al.  Differential protein accumulation in zeatin-and 2,4-D-treated cells of Catharanthus roseus. Correlation with indole alkaloid biosynthesis , 1993 .

[22]  D. Hochstrasser,et al.  Micropreparative two‐dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins , 1993, Electrophoresis.

[23]  Amos Bairoch,et al.  Human liver protein map: A reference database established by microsequencing and gel comparison , 1992, Electrophoresis.

[24]  C. Larsson,et al.  Introduction to the Plant Plasma Membrane — Its Molecular Composition and Organization , 1990 .

[25]  Yasuyuki Yamada,et al.  Characterization of Polypeptides that Accumulate in Cultured Nicotiana tabacum Cells , 1990 .

[26]  C. Stoscheck,et al.  Quantitation of protein. , 1990, Methods in enzymology.

[27]  M. Uemura,et al.  Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. , 1986, Plant physiology.