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Degradation of transplanted mitochondrial proteins by hepatocyte monolayers.

ABSTRACT: Reductively [3H]methylated rat mitochondria and mitochondrial-outer-membrane vesicles and mitochondrial-outer-membrane vesicles where monoamine oxidase is irreversibly labelled by [3H]pargyline have been transplanted into hepatocytes by poly(ethylene glycol)-mediated organelle or organelle-vesicle cell fusion. During subsequent culture of hepatocyte monolayers for 4-5 days, under conditions which mimic endogenous catabolic rates in vivo the transplanted organelle proteins retain their degradation characteristics observed in vivo (e.g. mitochondria: average t 1/2 72.5 h; monoamine oxidase: t1/2 55 h). In all cases protein degradation with first-order kinetics is only observed after an initial lag period (i.e. 24-30 h after fusion). Transplantation of fluorescein-conjugated organelles showed that the fluorescent material is rapidly internalized (average t1/2 1-6 h) and uniformly distributed in the cytoplasm. During a subsequent 18-24 h period (which corresponds to the lag period for intracellular destruction of transplanted mitochondrial material) the transplanted material is translocated to assume a perinuclear distribution. The destruction of transplanted mitochondrial proteins is compared with endogenous mitoribosomally synthesized proteins (average t1/2 52.5 h). Percoll fractionation of cell homogenates containing transplanted mitochondrial outer membranes where the enzyme monoamine oxidase is irreversibly labelled with [3H]pargyline shows a distribution of enzyme similar to lysosomal acid phosphatase. After transplantation of reductively methylated 3H-labelled mitochondrial-outer-membrane vesicles the cells were treated with leupeptin to alter lysosomal density. This treatment leads to the predominant association of acid phosphatase with dense structures, whereas the 3H-labelled transplanted material predominantly does not change density. Therefore transplanted mitochondrial-outer-membrane proteins are found in intracellular vesicular structures from which the proteins are donated for destruction, at least in part, by a lysosomal mechanism.

SUBMITTER: Evans PJ

PROVIDER: S-EPMC1152482 | biostudies-other | 1983 Oct

REPOSITORIES: biostudies-other

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Project description:1. The cholesteryl ester of isolated chylomicron-remnant particles was efficiently degraded by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors. 2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached saturation and conformed to a linear double-reciprocal plot. The V(max.) was determined as 80ng of cholesteryl ester/h per mg of protein and the apparent K(m) as 1.4mug of cholesteryl ester per mg of protein. The time course for the uptake and hydrolysis suggested that binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of native chylomicrons were degraded to a much smaller extent and their presence had only a small inhibitory effect on the degradation of chylomicron remnants. Intestinal very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to 50%, but had less influence on the amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake and hydrolysis, whereas d=1.21 infranatant had no effect. 4. The cholesteryl ester hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine. Only 28-36% of the unhydrolysed cholesteryl ester could be released from these cells by trypsin treatment, indicating that the major portion was truly intracellular. The particles that could be released from the cell surface by trypsin and those remaining in the medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant particles. Significant amounts of denser particles were thus not formed during contact with the cell surface. 5. The presence of heparin, as well as preincubation of the cells with heparin, increased the uptake of chylomicron remnants. This effect was most marked in the presence of serum. A much smaller proportion of the other serum lipoproteins was taken up, and this proportion was not increased by heparin.

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Degradation of transplanted mitochondrial proteins by hepatocyte monolayers.

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