Abstract
In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells. Upon treatment of the cells with BFA, RI332 becomes quantitatively O-glycosylated by Golgi glycosyltransferases that are transported back to the ER. Here we demonstrate that pretreatment of the cells with lovastatin, an inhibitor of HMG-CoA reductase, abrogates this modification and that mevalonate, the product formed in the step inhibited by the drug, is able to counteract the effect of lovastatin. We also show by immunofluorescence using mannosidase II as a Golgi marker that the BFA-induced retrograde transport of Golgi enzymes is blocked by lovastatin, although electron microscopy indicates that BFA causes disassembly of the Golgi apparatus into swollen vesicles and tubules. Our observations support the role of a prenylated protein, such as the geranylgeranylated small G protein Rab6, in the retrograde transport from the Golgi apparatus to the ER, since lovastatin acts by inhibiting its prenylation.
Highlights
A number of cytosolic proteins that are known to be involved in vesicular transport carry isoprenoid modifications required for their membrane attachment and biological activity
To ascertain that the effect of lovastatin is caused by the inhibitory action of this drug on hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cell cultures were pretreated with lovastatin together with an excess of mevalonate, the product normally formed in the step that is catalyzed by the enzyme, before performing pulse labeling and the chase incubations in the presence of brefeldin A (BFA)
We have demonstrated that the BFA-mediated retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) is inhibited by pretreatment of cells with lovastatin, while the addition of mevalonate counteracts the effect of lovastatin, indicating that mevalonate or downstream metabolites of mevalonate are required for this process
Summary
A number of cytosolic proteins that are known to be involved in vesicular transport carry isoprenoid modifications required for their membrane attachment and biological activity. We have previously shown that a truncated form of ribophorin I comprised of its 332 N-terminal amino acids (RI332) facing the lumen of the ER becomes a substrate for O-glycosylating enzymes that were redistributed to the ER through the action of BFA [11] Using this assay for retrograde Golgi to ER transport as well as immunofluorescence microscopy with Golgi markers, we could demonstrate that the back-transport of Golgi components to the ER requires calcium ions sequestered to intracellular stores [12]. We wanted to investigate whether BFA-mediated retrograde protein transport from the Golgi apparatus to the ER is compromised when the synthesis of isoprenoids is suppressed
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