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EnglishThe role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (less-than-or-equal-to 1000 Da) molecules. Vacuole formation was monitored from the uptake of I-125-tyramine-cellobiitol ((ITC)-I-125) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of (ITC)-I-125 was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-C-14]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTPgammaS (as well as GMP-PNP) decreased fractional rates of direct vacuolar (ITC)-I-125 uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation.
| Titolo: | De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs. |
| Autori: | |
| Data di pubblicazione: | 1994 |
| Rivista: | |
| Abstract: | The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (less-than-or-equal-to 1000 Da) molecules. Vacuole formation was monitored from the uptake of I-125-tyramine-cellobiitol ((ITC)-I-125) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of (ITC)-I-125 was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-C-14]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTPgammaS (as well as GMP-PNP) decreased fractional rates of direct vacuolar (ITC)-I-125 uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation. |
| Handle: | http://hdl.handle.net/11577/2488343 |
| Appare nelle tipologie: | 01.01 - Articolo in rivista |
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