Abstract
Past evidence has suggested that the lysosomal pathway is an important site of cytoplasmic RNA degradation in the hepatic parenchymal cell (Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14507-14519). We now provide additional support for this notion by quantitating degradable RNA in lysosomes and correlating its pool size with hepatic RNA degradation. Rat livers, previously labeled with [6-14C]orotic acid, were perfused with graded levels of amino acids over the full range of induced autophagy; RNA degradation was determined from [14C]cytidine release. Close correspondence between the marker beta-acetylglucosaminidase and the breakdown of RNA to cytidine in subcellular fractions indicated that the lysosome was the main site of catabolism, a conclusion supported by the fact that degradation was enhanced when external pH was lowered from 7 to 6. Although [14C]cytidine was also released in homogenates by the action of natural ribonucleases on cytosolic RNA, this source was eliminated by unlabeled exogenous RNA. The size of the degradable RNA pool in lysosomes, determined from the total release of cytidine in homogenates, correlated directly with rates of hepatic RNA degradation over the full range of basal and induced degradation. A direct correlation was also seen between RNA degradation and cytidine pools within lysosomal particles. Because cytosolic cytidine was not taken up by lysosomes under these conditions, the pool could only have arisen from the breakdown of intralysosomal RNA. As determined by cytidine production, these findings support the view that the lysosomal-vacuolar system is the main, if not sole, site of induced and basal RNA degradation in liver.
Highlights
From the Department of Cellular and Molecular Physiology, College of Medicine, The Pennsylvania StateUniversity, The Milton S
For example, that stringent amino acid on cytosolicRNA, this source was eliminated by unla-deprivationinthe perfused rat liverinduces identicalinbeled exogenousRNA
Because cytosolic cytidine ribosomes are prominent componeonfttshese vacuoles was not taken upby lysosomes under these conditions, in hepatocytes [9,10,11,12], and RNA breakdown is inhibitable by the pool could only have arisen from the breakdofwn intralysosomal RNA
Summary
After 40 min of single-pass perfusion with various amino acid additions, flow was switched to a second, cyclic perfusion reservoir containing 0.5 mM unlabeled cytidine to minimize label reutilization [5]. Liver (100-g rat), was computed by dividing the rate of label accumulation by the specific radioactivity of CMP in liver RNA [5]. This procedure, which has been fully validated for conditions in this study [5, 6], provides a measure of the rate of RNA breakdown to nucleoside end products that exists in liver cells at the moment cyclic perfusion is started. Separate control experiments revealed that [14C]cytidine added in trace amounts to unfractionated homogenates (37 “C, pH 7.0) was neither degraded nor rephosphorylated; losses during 150 min of incubation. Differences between means were evaluated by Student’s t test; values of p > 0.05 were considered not significant
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