Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [14C]2,4-dichlorophenoxyacetic acid and [3H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [3H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [14C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

[1]  K. Palme,et al.  An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: identification by photoaffinity labeling and purification of a 23-kda polypeptide. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[2]  L. Staehelin,et al.  Effect of Brefeldin A on the Structure of the Golgi Apparatus and on the Synthesis and Secretion of Proteins and Polysaccharides in Sycamore Maple (Acer pseudoplatanus) Suspension-Cultured Cells , 1993, Plant physiology.

[3]  K. Palme,et al.  5'-Azido-[3,6-3H2]-1-napthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: identification of a 23-kDa protein from maize coleoptile plasma membranes. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Rothman,et al.  Mechanisms of intracellular protein transport , 1994, Nature.

[5]  M. Goldsmith,et al.  The Polar Transport of Auxin , 1977 .

[6]  P. Guerrier,et al.  6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity. , 1988, Experimental cell research.

[7]  J. Reagan,et al.  The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein , 1996, Plant physiology.

[8]  J. Guern,et al.  Involvement of protein phosphorylation in the early steps of transduction of the oligogalacturonide signal in tobacco cells , 1996 .

[9]  K. Feldmann,et al.  Arabidopsis AUX1 Gene: A Permease-Like Regulator of Root Gravitropism , 1996, Science.

[10]  C. Hawes,et al.  The distribution of secretory products in plant cells is affected by brefeldin A , 1993 .

[11]  J. Guern,et al.  Regulation of intracellular pH in plant cells , 1991 .

[12]  R. Miller,et al.  Nutrient requirements of suspension cultures of soybean root cells. , 1968, Experimental cell research.

[13]  John C. Walker,et al.  PLANT PROTEIN PHOSPHATASES. , 1996, Annual review of plant physiology and plant molecular biology.

[14]  P. Kaufman,et al.  Hormones and the Orientation of Growth , 1987 .

[15]  B. Pickard.  Roles of Hormones in Phototropism , 1985 .

[16]  C. MacKintosh,et al.  Protein phosphatase inhibitors activate anti‐fungal defence responses of soybean cotyledons and cell cultures , 1994 .

[17]  G. Hicks,et al.  Specific auxin-binding proteins in the plasma membrane: receptors or transporters? , 1992, Biochemical Society transactions.

[18]  D. Morris,et al.  Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments , 1991 .

[19]  B. Pickard.  Roles of Hormones, Protons and Calcium in Geotropism , 1985 .

[20]  S. Thomine,et al.  Cytosolic protons as secondary messengers in elicitor-induced defence responses. , 1994, Biochemical Society symposium.

[21]  D. Söll,et al.  A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis. , 1996, The EMBO journal.

[22]  R. M. Spanswick,et al.  Auxin transport in suspension-cultured soybean root cells : I. Characterization. , 1991, Plant physiology.

[23]  B. Satiat-Jeunemaitre,et al.  Brefeldin A effects in plant and fungal cells: something new about vesicle trafficking? , 1996, Journal of microscopy.

[24]  P. Rubery.  Phytotropins: receptors and endogenous ligands. , 1990, Symposia of the Society for Experimental Biology.

[25]  G. Muday,et al.  NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton. , 1994, The Plant cell.

[26]  T. Caspari,et al.  MEMBRANE TRANSPORT CARRIERS. , 1996, Annual review of plant physiology and plant molecular biology.

[27]  P. Bernasconi.  Effect of synthetic and natural protein tyrosine kinase inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls , 1996 .