Abstract
The molecular mechanisms involved in the degradation of individual cellular proteins are probably unique and characteristic. We have investigated in rat liver the degradation of glyceraldehyde-3-phosphate dehydrogenase, an abundant cytosolic enzyme of the glycolytic pathway. Immunoblot analysis of isolated liver lysosomes from rats treated with lysosomal inhibitors show that this protein is degraded, at least in part, by a lysosomal pathway. This pathway was further investigated by incubating the enzyme with lysosomes in a cell-free system, followed by proteolysis measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysosomes, and electron microscopic immunocytochemistry. We postulate that the degradative mechanism of glyceraldehyde-3-phosphate dehydrogenase includes a temperature-dependent lysosomal pathway, different from classical nonspecific macroautophagy. The postulated pathway involves: binding of the enzyme to the lysosomal membrane, entry into the lysosomal matrix, and degradation. This cell-free system, which can also incorporate in vitro synthesized proteins, should allow further advances toward clarifying the complex signals that regulate protein degradation as well as its close interrelationship with protein synthesis.
Highlights
Genase includes a temperature-dependent lysosomal To this aim,we used immunoblot analysisof isolated lysosopathway, different from classical nonspecific macroau- mal fractions as well as an i n uitro model of lysosomal degtophagy
Intracellular proteins in mammaliancells are continuously Materials-GAPDH, glutamate dehydrogenase being degraded at different rates
The observed complexity could reflect the fact that the mocatalase, PstI, and BamHI were from Boehringer Mannheim (Germany). 3-Phosphoglyceric phosphokinase, aldolase, phosphoglycerate mutase, L-lactic dehydrogenase, malic dehydrogenase, cytoplasmic and mitochondrial, albumin, trypsin, chloroquine, leupeptin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, chymostatin, o-phenantroline, E-64, lecular mechanism involved in the degradatioonf each partic- Triton X-100, oso4, uranyl acetate, MES, and citric acid were from ular protein is unique, characteristic, and different from oth- Sigma
Summary
VOl. 268, No 14, Issue of May 15, pp. 10463-10470,1993 Printed in U.S.A. Uptake and Degradationof Glyceraldehyde-3-phosphate Dehydrogenase by RatLiver Lysosomes*. Immunoblotanalysis of isolated liver oxidative stepin glycolysis, itis very abundant,andits lysosomes from rats treated with lysosomal inhibitors i n uitro inactivation mechanisms havebeen extensively studshow that this protein is degraded, at least inpart, by a lysosomal pathway. This pathway was further investigated by incubating the enzyme with lysosomes in a cell-freesystem, followed by proteolysis measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysosomes, and electron microscopic immunocytochemistry. The postulated pathway involves: binding of radation based on onepreviously described [24,25,26,27], which we the enzyme to the lysosomal membrane, entry intothe have further evolved with procedures employed to follow the lysosomal matrix, and degradation This cell-free sys- traffic of intracellular proteins to othecrell organelles.
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