Temperature-modulation of protein phosphorylation in cell-free extracts of alfalfa

• Main Author: Labbé, Etienne.
• Other Authors: Dhindsa, R. S. (advisor)
• Format: Others
• Language: en
• Published: McGill University 1996
• Subjects: Alfalfa > Effect of cold on.; Cold adaptation.; Phosphorylation.; Phosphoproteins.
• Online Access: http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=20151

The effects of temperature on a 58-kDa phosphoprotein (PP58) have been examined in cell-free extracts of two alfalfa (Medicago sativa L.) cultivars, Apica and Trek. In the extracts prepared without the use of Triton X-100, PP58 is present in a 12,000 x g (P12), 28,000 x g (P28) and 100,000 x g (P100) pellets but is enriched in the P28 fraction. In these fractions PP58 is substantially and equally phosphorylated at both 4° and 24°C. When extracts are prepared in the presence Triton X-100, PP58 is present in the 28,000 x g supernatant (TXS fraction) is extensively dephosphorylated at 24°C but highly phosphorylated at 4°C. The phosphorylation of this protein increased sharply as temperature declined below 12°C, and was 15 times greater at 0° than at 24°C. The phosphorylation level doubled between 12° and 8°C and again between 8° and 4°C. Thus temperature effect is not mediated by Q10 effect. Interestingly, temperature-response curve of PP58 phosphorylation is similar to that of the reported cold-induced calcium influx (Plant Cell 7: 321-331). Labeling reactions carried out in the presence of [gamma-35S]thioATP indicated that low temperature inhibited the dephosphorylation reaction. These results were not mimicked at room temperature by the protein phosphatase 1 and 2A inhibitor okadaic acid. In reactions performed at 4°C, addition of calcium caused a 2-fold increase in the phosphorylation of PP58. A decrease in phosphorylation was observed when equimolar amounts of EGTA were added in the presence of MgCl 2 or MnCl2, but not in the presence of CaCl2, suggesting that this protein is phosphorylated by a calcium-dependent protein kinase. These results are consistent with the suggestion that PP58 and its putative kinase are membrane-localized whereas the putative PP58 phosphatase is a loosely-associated membrane peripheral protein lost to the supernatant during fractionation. We suggest that PP58 could be involved in low te