Abstract
The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.
Highlights
Chaperones resident to the endoplasmic reticulum (ER)1 catalyze the folding of nascent polypeptides to tertiary structures that are competent for ER to Golgi transport
In cell types overproducing proteins that translocate to the ER but that are incapable of ER to Golgi transport, GRP78 is chronically elevated, and mRNA translation is sustained upon challenge with ER stressors [6]
Modification of GRP78 in Response to the Inhibition of mRNA Translation by Various Agents—Good evidence supports the hypothesis that GRP78 functions in part to coordinate the respective rates of ER protein processing and mRNA translation [6]
Summary
Chaperones resident to the endoplasmic reticulum (ER) catalyze the folding of nascent polypeptides to tertiary structures that are competent for ER to Golgi transport. GRP78 is reported to undergo post-translational modification by mono-ADP-ribosylation and by phosphorylation [15, 16]. The modification of GRP78 is suppressed by conditions that inhibit glycoprotein processing within the ER such as glucose depletion or treatments with tunicamycin, glucose analogs, or Ca2ϩ ionophores [15, 19, 21, 22]. The unmodified, unbound form of GRP78 is thought, to function as the active form of the chaperone that is available to interact with processing intermediates derived from co-translational translocation. It is this form of the chaperone that must be subject to inactivation through covalent modification. This paper is available on line at http://www.jbc.org tional ADP-ribosylation of GRP78 and the degree of eIF-2 phosphorylation that was explicable in terms of changing contents of the active monomer
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