Regulation of lysosomal fusion during deprivation-induced autophagy in perfused rat liver

Abstract

Previous studies have demonstrated that autophagic vacuoles induced in hepatocytes of the perfused rat liver can be effectively separated from secondary lysosomes in self-generating gradients of colloidal silica/polyvinylpyrrolidone. Here we show that, as autophagy is increased by graded amino acid deprivation, the lysosomal marker N-acetyl-beta-D-glucosaminidase shifts progressively from dense to buoyant gradient fractions, an effect attributable to enhanced lysosome-autophagosome fusion and the formation of additional autolysosomes. Both the amount of the shift and the aggregate volume of autolysosomes were directly proportional to the increase in protein degradation above basal. Because the total quantity of protein sequestered in the vacuoles is also a direct function of deprivation-induced proteolysis, the relative specific activity of the marker enzyme in autolysosomes (enrichment) would be expected to remain constant over the full range of deprivation; numerical estimates of enrichment, in fact, did not differ from the mean (43.9) by more than 5%. The close correspondence between cytoplasmic sequestration and fusion was maintained, despite a greater than 3-fold increase in the average volume of individual vacuoles. This suggests the existence of an intrinsic regulatory mechanism which limits the number of fusions per vacuole, on the basis of information derived from vacuolar volume.

Selective Uptake of Cytosolic, Peroxisomal, and Plasma Membrane Proteins into the Yeast Lysosome for Degradation

“…Most short-lived normal proteins and abnormal proteins are degraded in the cytosolic, ubiquitin-dependent, or proteasome-dependent pathway (1-3), whereas most long-lived proteins are degraded in lysosomes (4,5). Lysosomal degradation of cytosolic proteins is regulated by hormones and growth factors and is increased when cells are starved of nutrients (4 -6).…”

Inhibition of intracellular protein degradation by ethanol in perfused rat liver

“…Because the shifts are measurements of dense-body fusion and, hence, indicate the total quantity of induced autophagy, a suppression of sequestration would be accompanied by a marker shift into the dense fractions. On the other hand, lysosomotropic inhibition is frequently associated with an increase in overt autophagy (Furuno et al, 1982;Kovaics et al, 1982;Kominami et al, 1983;Glaumann et al, 1986) and would be expected to produce a shift in the opposite direction (Surmacz et al, 1987). Surprisingly, the results of gradient studies revealed that ethanol did not have any effect on movement of the marker.…”

“…It could also explain the suppression at 0 x , but, since most of the increase in protein degradation that occurs in the absence of amino acids is the consequence of induced autophagy (Schworer et al, 1981), one must exclude other possible effects of ethanol on autophagy as well. Previous studies with colloidal-silica/PVP gradients have shown that decreases in deprivation-induced rates of proteolysis resulting from lysosomotropic inhibition can readily be differentiated from effects on autophagic sequestration by shifts in lysosomal marker away from or into gradient fractions containing dense lysosomes (Surmacz et al, 1987). Because the shifts are measurements of dense-body fusion and, hence, indicate the total quantity of induced autophagy, a suppression of sequestration would be accompanied by a marker shift into the dense fractions.…”

“…Taken together, Table 3 clearly support the conclusion that the effect on protein degradation with ethanol or 0 x is mediated by two mechanisms: (i) a decrease in autophagic sequestration and (ii) lysosomotropic inhibition. The deviation in the autophagosome/autolysosome ratio is particulary compelling in this regard, because the proportionality between these two populations has always been extremely close (Schworer et al, 1981;Surmacz et al, 1987). There are several lines of evidence which indicate that protein degradation rather than synthesis is the dominant site for regulating the cytoplasmic protein content of the hepatic parenchymal cell (Mortimore & Poso, 1984;Scornik, 1984).…”

Nutritional regulation of the anabolic fate of amino acids within the liver in mammals: concepts arising fromin vivostudies

“…However, stimulation of protein synthesis by feeding alone cannot explain the high lability of liver protein content observed during food deprivation (16,78,79) . This suggests that the constitutive liver protein pool in catabolic states is primarily regulated by protein degradation (78,80,81) . Hepatic protein synthesis (measured as albumin synthesis) is also responsive to dietary protein intake (82) .…”