J. Biol. Chem., Vol. 263, Issue 18, 8759-8764, 06, 1988
Dissection of membrane protein degradation mechanisms by reversible inhibitors
JF Hare
Department of Biochemistry, School of Medicine, Oregon Health Sciences University, Portland 97201.
The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma
membrane polypeptides in response to reversible temperature and
lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were
radiolabeled and left for 24 h to allow the removal of rapidly degraded
proteins. Remaining trichloroacetic acid-precipitable protein was degraded
(t 1/2 = 40-68 h) by an apparent first order process 60- 86% sensitive to
10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by
temperature reduction to 18 degrees C. Thus, membrane proteins are selected
for degradation in a time-dependent manner by a system which is sensitive
to both 18 degrees C and to lysosomotropic amines. When inhibitory
conditions were removed after 40-48 h, degradation of 125I-labeled protein
resumed at the same rate as that seen in their absence. Since membrane
proteins do not exhibit accelerated degradation after removal of inhibitory
conditions, there can be no marking or sorting of those proteins destined
for degradation during the 40-h exposure to inhibitory conditions. Exposure
to amines or 18 degrees C did not affect the position of two-dimensionally
resolved labeled polypeptides. Fractionation of labeled cells on Percoll
gradients after 40 h of exposure to low temperature or amines showed that
labeled protein remained in the plasma membrane fractions of the gradient
although shifted to a slightly lower buoyant density in the presence of
amines. These results support the notion that selection of plasma membrane
proteins for degradation requires their internalization into acidic
vesicles. Lysosomotropic amines and reduced temperature interfere with the
selection process by preventing membrane fusion events.
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