The Effect of Sterols and Brefeldin A on Protein Degradation in UT-1 Cells

Abstract

UT-1 cells, a mutant Chinese hamster ovary (CHO) cell line induced to produce an abundance of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), were used to determine the effects of sterols and brefeldin A on the degradation of this enzyme. Brefeldin A has been shown to cause retention of proteins in and relocation of proteins to the endoplasmic reticulum (ER). UT-1 cells were incubated with (a) sterols only (12 μg/ml cholesterol and 0.2 μg/ml 25-hydroxycholesterol), (b) sterols and brefeldin A (0.5 μg/ml), and (c) brefeldin A only. Western blot analysis showed that incubation with sterols and brefeldin A decreased HMGR levels more slowly than incubation with sterols alone over the first 24-36 h of incubation; however, the rates were not significantly different. By 48 h of incubation, HMGR had decreased to a level comparable to that found when cells were incubated in sterols only. Incubation with brefeldin A alone did not cause a decrease in HMGR over the same 48-h time period. HMGR was undetectable in parental CHO cells under all of the conditions described. Indirect immunofluorescence microscopy revealed a pattern of tight, perinuclear staining with sterol incubation. After 48 h in sterols, HMGR staining was uniformly decreased throughout the cytoplasm. This change in staining pattern is also observed during incubation of UT-1 cells with sterols and brefeldin A. Incubation for 48 h with brefeldin A alone had no effect on the tight perinuclear pattern originally observed. Diffuse, faint staining of CHO cells under all conditions served as a negative control. The results of these experiments indicated that brefeldin A, and therefore retention of proteins in the ER, does not interfere with the degradation of HMG CoA reductase. Despite the presence of brefeldin A, sterol-mediated dispersal and degradation of the crystalloid ER (CER) continued in UT-1 cells. Lack of brefeldin A sensitivity implied that the mechanism for CER dissolution was distinct from previously described mechanisms for ER to Golgi transport.