Abstract
Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.
Highlights
(ASOR) mediated by the galactosyl receptorin rat actosyl receptors are reutilized in a cyclic fashion, which does hepatocytes is a cyclic process.1a51-ASOR-receptor not require protein synthesis
Sincae true cycle has a ther- be a requirement for the input of additional external energy modynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle
Through discontinuous Percoll gradients, and only vi- For the purpose of this study, we initially viewed endocyable cells were isolated and incubated at 37 OC to ini- tosis mediated by the galactosyl receptor as a three-part tiate a synchronous single round of endocytosis
Summary
Continuous endocytosis of ‘aaI-asialo-orosomucoid (galactosyl) receptors within the cell. The endocytic process is presently thought to surface-bound ligand was unaffected until the ATP result from the closing of a coated pit to form a coated vesicle levels decreased to 30%or less; at >98%ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. The coated vesicles presumably undergo maturation and/or translocation processes that include sloughing the clathrin coat, which requires ATP in uitro ( l l ) , fusion of primary endosomes to form secondary endosomes and tubules, and eventually fusion with lysosomes [6,12,13] During these processing steps the receptor must be dissociated and segregated from the ligand and returned to thecell surface. A preliminary report of these results has been presented [23]
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