No abstract

SUMMARYHeat stress induces an array of physiological adjustments that facilitate continued homeostasis and survival during periods of elevated temperatures. Here, we report that within minutes of a sudden temperature increase, plants deploy specific phospholipids to specific intracellular locations: phospholipase D (PLD) and a phosphatidylinositolphosphate kinase (PIPK) are activated, and phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) rapidly accumulate, with the heat-induced PIP 2 localized to the plasma membrane, nuclear envelope, nucleolus and punctate cytoplasmic structures. Increases in the steady-state levels of PA and PIP 2 occur within several minutes of temperature increases from ambient levels of 20-25°C to 35°C and above. Similar patterns were observed in heat-stressed Arabidopsis seedlings and rice leaves. The PA that accumulates in response to temperature increases results in large part from the activation of PLD rather than the sequential action of phospholipase C and diacylglycerol kinase, the alternative pathway used to produce this lipid. Pulse-labelling analysis revealed that the PIP 2 response is due to the activation of a PIPK rather than inhibition of a lipase or a PIP 2 phosphatase. Inhibitor experiments suggest that the PIP 2 response requires signalling through a G-protein, as aluminium fluoride blocks heat-induced PIP 2 increases. These results are discussed in the context of the diverse cellular roles played by PIP 2 and PA, including regulation of ion channels and the cytoskeleton.

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/ BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/ BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.

We have detected a proteolytic mechanism in chloroplasts that selectively and rapidly degrades the imported small subunit of ribulose 1,5-bisphosphate carboxylase when pools of the chloroplast-synthesized large subunit are depleted. This degradation system is constitutively present and appears to be responsible for precise stoichiometric accumulation of the two subunits of the enzyme. We believe similar proteolytic mechanisms participate in regulating the accumulation of other photosynthetic proteins during chloroplast biogenesis.Ribulose 1,5-bisphosphate carboxylase/oxygenase [RbuP2Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the major protein in C3 plants, is localized in the soluble fraction of chloroplasts, where it catalyzes the initial steps in CO2 reduction (1, 2). The RbuP2Case holoenzyme consists of eight copies of each of the 52,000-to 56,000-dalton large subunit and the 12,000-to 15,000-dalton small subunit. Much is known concerning the mode of synthesis of the large subunit within chloroplasts and of the nuclear-encoded small subunit in the cytoplasm. However, many aspects of the regulation of synthesis and the pathway for subunit assembly remain to be elucidated. In particular, the mechanism(s) by which stoichiometric amounts of large and small subunits are produced during chloroplast biogenesis have not been established. In this paper, we provide evidence that, when the small subunit is produced in excess of the large, it is selectively and rapidly degraded by a proteolytic activity that is constitutively present in chloroplasts. MATERIALS 43 Ci/mg at 100% isotopic enrichment; 1 Ci = 37 GBq; ICN Chemical and Radioisotope Division) was added to a final concentration of 1 mCi/ml. The chase periods were begun on addition of a 1/10th vol of 100 mM Na2SO4. At intervals, 100-,ul aliquots were transferred to 900 ul of 100% acetone at 0°C to terminate protein synthesis and degradation. Electrophoresis. Acetone precipitates were dissolved in 2% NaDodSO4/60 mM Tris HCI, pH 8.6/60 mM dithiothreitol/5 mM E-aminocaproic acid/i mM benzamidine/15% (wt/vol) sucrose, and 5-1Al aliquots were removed for radioactivity determination (4). Equal amounts of labeled protein were subjected to electrophoresis in 10-20% (wt/vol) gradient polyacrylamide gels prepared in the buffer system of Laemmli (5). For two-dimensional electrophoresis, the precipitates were dissolved in 2% Ampholines (pH 3.5-10; LKB)/2% Nonidet P-40 (Particle Data Laboratories)/10 mM dithiothreitol/9.5 M urea/10 mM methylamine and subjected to nonequilibrium pH gradient electrophoresis for 1,600 V-hr as described (6), except that the formulation of ampholytes for a pH range of 3.5 to 9.5 (7) was used. Electrophoresis in the second dimension was carried out in the NaDodSO4/polyacrylamide gradient gels described above. Gels were treated for fluorography (8) before exposure to Kodak X-Omat AR film at -70°C. RESULTSTo study the in vivo events involved in the synthesis of RbuP2Case, we have used Chlamydomonas in pulse-chase ...