Intracellular protein catabolism: evidence for sequestration of proteins into an intermediate-filament fraction before lysosomal degradation.
Iodinated [125I] glycolytic enzymes (lactate dehydrogenase, pyruvate kinase and glyceraldehyde phosphate dehydrogenase) and bovine serum albumin have been erythrocyte microinjected or scrape loaded into confluent 3T3-L1 cells. The glycolytic enzymes are rapidly (within 30 min) sequestered into a form which is inextractable with sequential treatments with digitonin, Triton X-100 and potassium iodide. Bovine serum albumin is readily extractable by digitonin. Glycolytic enzymes are degraded relatively slowly (t1/2's 124-330 h) by a lysosomal mechanism inhibitable by NH4Cl (50-91%), 3-methyl adenine (62-70%) and leupeptin (47-80%). Bovine serum albumin is degraded relatively rapidly (t1/2 17-20 h) by a non-lysosomal mechanism. Nycodenz density gradient fractionation of homogenates of 3T3-L1 cells after microinjection shows that [125I]-lactate dehydrogenase sediments in a dense fraction containing nuclei (DNA) and lacking cytosolic, lysosomal, plasma membrane or mitochondrial marker enzymes. The [125I]-enzyme is not released from this fraction by the sequential detergent/salt fractionation described above. Analysis of the detergent/salt inextractable residue by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol shows nuclear histones and vimentin. [125I]-lactate dehydrogenase does not enter the polyacrylamide gel in the absence of 2-mercaptoethanol. Importantly endogenous proteins are found in the residue in the presence of leupeptin. The data suggest that sequestration of proteins into a nuclear-intermediate filament fraction may precede lysosomal degradation. Iodinated [125]-Sendai virus HN and F membrane glycoproteins in reconstituted Sendai virus envelope (RSVE) have been fused with growing HTC cells. HN and F are rapidly internalised (within 5 h) to assume a perinuclear distribution before lysosomal degradation. HN and F proteins are either extractable predominantly with triton X-100 or are inextractable by the treatments described above. Nycodenz fractionation of HTC cell homogenates after RSVE-cell fusion shows that HN and F sediment predominantly as a dense peak, enriched in DNA and free from the markers indicated above, and a less dense peak enriched in lysosomal marker enzymes. HN and F proteins translocate from the dense to the less dense peak and are degraded lysosomally (t1/2 av. 70 h). Leupeptin slows the translocation. The detergent/salt inextractable residue is enriched in nuclear histones and vimentin. The data suggest that Sendai viral membrane glycoproteins are sequestered into a nuclear-intermediate filament fraction before lysosomal degradation.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005057:Eukaryotic Cells / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008845:Microinjections; D010455:Peptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*
D006023:Glycoproteins; D008565:Membrane Proteins; D010455:Peptides; D011506:Proteins; D007770:L-Lactate Dehydrogenase