Minisymposium 24: Temperature Response

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Abs # M2401: Heat Shock-Induced Changes in Lipid and Protein Metabolism in the Endoplasmic Reticulum of Barley Aleurone Layers - An ER Stress Response

Presenter:	Brodl, Mark R.       Contact Presenter
Authors	Brodl, Mark R. (B)   Johnston, Mark K (A)   Jacob, Nitya  (C)
Affiliations:	(A): Knox College, Department of Biology (B): Trinity University, Department of Biology (C): Oxford College of Emory University, Department of Biology

Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing secretory protein mRNAs. In addition, the ER membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted by either "ramping" to heat shock temperature over 3 to 6 hours or maintaining aleruone layers at warmer temperatures (but not so warm as to induce HSP synthesis) prior to heat shock. The ER membranes of heat-adapted cells have higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells, and secretory proteins are synthesized at heat shock temperature. To better understand heat shock-induced changes in aleruone layer ER, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock induced significantly the activity of phospholipases A2 and D within 30 min of heat shock. Shortly thereafter lipase induction (within 60 min of the onset of heat shock), significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. In examining the integral membrane proteins of the ER, heat shock induced only minor changes in protein composition as observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes (purified on continuous sucrose gradients and then ultrasonicated to release lumenal proteins) relative to protein, causing heat-shocked ER to migrate at higher surcose densities on continuous gradients. Intact aleurone cells were ultracentrifuged, fixed and sectioned then examined by light and transmission electron microscopy, and the ER band in these in vivo fractionated cells appeared to increase in density. Taken together, the changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell.