Abstract
When barley (Hordeum vulgare) aleurone layers are subjected to heat shock there is a selective degradation of the normally stable mRNAs encoding secreted proteins. Messages for nonsecreted proteins are not degraded. The synthesis of heat shock proteins is not required for this selective message degradation. Our hypothesis explaining this phenomenon is that a component of the early steps in the synthesis of secreted proteins is damaged by heat shock, resulting in a selective halt in translation on secretory mRNAs, which may in turn lead to degradation of those messages. The first committed step in the synthesis of secreted proteins is the binding of the nascent signal sequence to the signal recognition particle. We have obtained cDNA clones and antibodies for the barley 54-kDa subunit of the signal recognition particle. In cell fractionation experiments, more signal recognition particle was bound to the endoplasmic reticulum membranes and less was in the free particle fraction following a heat shock. The results suggest that heat shock inhibits the release of the signal recognition particle from the endoplasmic reticulum. This would, in turn, inhibit the resumption of translation and may be the underlying cause of the secretory message degradation.
Highlights
When organisms are subjected to the stress of high temperature, synthesis of a set of new proteins, the heat shock proteins,1 is induced
The results suggest that heat shock inhibits the release of the signal recognition particle from the endoplasmic reticulum
Based on our hypothesis regarding the effect of heat shock on the secretory system, we considered the possibility that heat shock may inhibit the binding of signal recognition particle (SRP) to the ER membranes
Summary
Probes used for the RNA gel blots were barley srp cDNA, barley ␣-amylase high pI cDNA CDNA Library Construction, Screening, and Clone Characterization—A barley leaf gt cDNA library, prepared from RNA extracted from 7- to 10-day old seedlings, was provided by Dr R. The library was screened with the A. thaliana srp cDNA fragment and, at high stringency, with the 1.1-kilobase barley clone. Powdered samples were thawed in buffer (200 mM Tris, pH 8.5, 30 mM MgCl2, 50 mM KCl, 350 mM sucrose, 6 mM 2-mercaptoethanol) containing an RNase/protease inhibitor mixture [30], filtered through Miracloth, spun at 15,000 ϫ g at 4 °C for 15 min to pellet cell debris. RNA blot analyses indicated that RNA isolated from the samples was the size expected for ␣-amylase mRNA
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