Abstract
We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that alpha-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of alpha-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of beta-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on beta-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far.
Highlights
Synthesized secretory proteins are transported from the endoplasmic reticulum (ER)1 to the cell surface via the
In the present study we examined the effect of Nordihydroguaiaretic acid (NDGA) on secretion and intracellular processing of secretory proteins, confirming that the drug blocks the protein transport from the ER to the Golgi complex
Albumin and C3 are initially synthesized as proforms, which are converted into mature forms at the trans-Golgi network (TGN) [5, 11]
Summary
The membrane was incubated with rabbit anti--COP IgG (25 g/ml) for 1 h, followed by incubation with peroxidase-conjugated anti-rabbit IgG for 1 h. Cells were briefly washed with PBS and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. The fixed cells were washed, permeabilized with 0.1% saponin in PBS and incubated with the indicated primary antibodies for 15 min, followed by incubation for 15 min with fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. The HepG2 cells were incubated for 2 h with anti-albumin antibodies, followed by incubation for 1 h with peroxidase-conjugated goat anti-rabbit IgG. The H35P15 cells were incubated with anti-Man II antibodies and with biotinylated anti-mouse IgG and peroxidase-conjugated streptavidin for 1 h each. The cells were processed for electron microscopy as described previously [12]
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