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(Received for publication, June 26, 1996, and in revised form, February 19, 1997)
From the Department of Medicine, College of Physicians and Surgeons
of Columbia University, New York, New York 10032
Newly synthesized
apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi
compartment in HepG2 cells. A major site of this early degradation
seems to be on the cytosolic side of the endoplasmic reticulum (ER)
membrane and is sensitive to
N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which can
inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates
translocation of nascent apoB across the ER membrane, also reduces
early degradation. In the present studies, we have used brefeldin A
(BFA), which inhibits vesicular transport from the ER to the Golgi, to
demonstrate that apoB can also be degraded by an ER luminal proteolytic
activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were co-treated with ALLN, which protects apoB
but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA
or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an
ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with
dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was
inhibited. Furthermore, addition of DTT resulted in the accumulation of
a 70-kDa amino-terminal fragment of apoB. Both full-length and
amino-terminal apoB were degraded if DTT was removed from the
incubation media; both were secreted if only BFA was removed. Thus,
even after apoB is translocated into the ER lumen (thereby avoiding the
initial proteolytic pathway), it can potentially be degraded by a
lumenal proteolytic process that is ALLN-resistant but DTT-sensitive.
The present results, together with previous studies, suggest that at
least two distinct steps may be involved in the posttranslational
degradation of apoB: 1) the first occurs while apoB is partially
translocated and is ALLN-sensitive; and 2) the second occurs in the ER
lumen and is DTT-sensitive. Finally, our results support the hypothesis
that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a
DTT-sensitive pathway.
Volume 272, Number 17,
Issue of April 25, 1997
pp. 11575-11580
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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