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A two-site model for ApoB degradation in HepG2 cells
1Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
The Journal of Biological Chemistry
|April 25, 1997
Summary
Newly synthesized apolipoprotein B (apoB) is rapidly degraded in the endoplasmic reticulum (ER) via two distinct pathways. One pathway is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), while the other, occurring in the ER lumen, is sensitive to dithiothreitol (DTT).
Area of Science:
- Cell Biology
- Biochemistry
- Proteostasis
Background:
- Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in HepG2 cells.
- Early degradation occurs on the cytosolic side of the ER membrane and is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN).
- Oleate (OA) treatment reduces early apoB degradation by facilitating ER translocation.
Purpose of the Study:
- To investigate apoB degradation pathways in the endoplasmic reticulum (ER).
- To differentiate between ALLN-sensitive and other proteolytic activities.
- To elucidate the role of ER luminal proteolysis in apoB degradation.
Main Methods:
- Utilized brefeldin A (BFA) to inhibit ER-to-Golgi transport.
- Co-treated cells with ALLN, OA, and dithiothreitol (DTT).
- Analyzed apoB degradation and fragment generation using biochemical assays.
Main Results:
- BFA revealed an ALLN-resistant, ER luminal proteolytic activity.
- OA enhanced apoB translocation into the ER lumen, exposing it to this ALLN-resistant pathway.
- DTT inhibited apoB degradation in the ER lumen and led to the accumulation of a 70-kDa amino-terminal fragment.
Conclusions:
- At least two distinct posttranslational degradation steps for apoB exist: an ALLN-sensitive step during partial translocation and a DTT-sensitive step in the ER lumen.
- Degradation of partially translocated apoB generates a 70-kDa fragment degraded via a DTT-sensitive pathway in the ER lumen.