Degradation of transplanted mitochondrial proteins by hepatocyte monolayers.
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.
D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D007425:Intracellular Membranes / Q000378:metabolism / Q000637:transplantation; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000378:metabolism* / Q000637:transplantation; D008995:Monoamine Oxidase / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism; D013367:Submitochondrial Particles / Q000378:metabolism
D008565:Membrane Proteins; D011506:Proteins; D012269:Ribosomal Proteins; D008995:Monoamine Oxidase