A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with transplanted Sendai-viral envelope proteins in HTC cells

Abstract

Reconstituted Sendai-viral envelopes (RSVE) were fused with hepatoma tissue-culture (HTC) cells, thereby introducing viral membrane glycoproteins into the plasma membrane [Earl, Billett, Hunneyball & Mayer (1987) Biochem. J. 241, 801-807]. Fractionation of homogenized cells on Nycodenz gradients shows that much of the viral 125I-labelled HN and F proteins were rapidly sequestered into a dense fraction distinct from fractions containing plasma membrane, lysosomes and mitochondria. Electron microscopy (results not shown) indicates that the dense fraction contains nuclear residues, multivesicular structures, dense bodies and fibrous structures. Both the dense fraction and a hexosaminidase-enriched fraction contain trichloroacetic acid-insoluble radioactivity, including intact 125I-labelled viral proteins. The viral proteins are progressively transferred from the dense fraction to the hexosaminidase-enriched fraction; the transfer is retarded by 50 micrograms of leupeptin/ml. Trichloroacetic acid-soluble radiolabel is progressively released into the culture medium as the proteins are degraded. Within 5 h after transplantation of viral HN and F proteins into recipient cells, a proportion (approx. 45%) of the 125I-labelled glycoproteins cannot be extracted by sequentially treating cells with digitonin (1 mg/ml), Triton X-100 (1%, w/v) and 0.3 M-KI. HN and F proteins in the non-extractable residue are tightly associated with nuclear-intermediate-filament (vimentin) material, as shown by Western blots and electron microscopy. The viral proteins are progressively transferred out of the nuclear-intermediate-filament residue; the transfer is slowed when cells are cultured with leupeptin. The data are consistent with the notion that transplanted viral HN and F proteins are sequestered to a perinuclear site in tight association with intermediate filaments before transfer into the autophagolysosomal system for degradation.