Internalization, intracellular sorting, and recycling of cell surface sialoglycoproteins in HeLa cells

Author

Jordan Barry Fishman

Date of Award

6-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

John S. Cook

Committee Members

Stephen Kennel, Salil Niyogi, Fred Snyder

Abstract

Cell surface sialic acid residues were specifically modified to a radiolabeled, neuraminidase-sensitive, 7-carbon analog ([³H]-AcNeu7) by sequential treatment of HeLa or HTC cells with NaBH₄, NalO₄, and NaBC[³H]₄. [³H]-AcNeu7 is neither reutilized nor is it metabolized by intact cells. With time (approx. 30 hours) cell surface [³H]-AcNeu7-glycoconjugates equilibrate with the intracellular milieu. Approximately 30% of the cell associated radioactivity is then sensitive to hydrolysis by added extracellular neuraminidase. Cells allowed to internalize cell surface [³H]-AcNeu7-glycoconjugates and subsequently exposed to neuraminidase to remove surface radioactivity contain an intracellular pool with a relatively high specific radioactivity compared to the plasma membrane. Resuspension of these cells in growth medium and assay of neuraminidase-sensitive radioactivity at various times thereafter show a return of labeled glycoconjugates to the cell surface again reaching euqilibrium with approximately 30% of the cell-associated radioactivity sensitive to neuraminidase. After labeling, approximately 16 labeled sialoglycoproteins could be identified by SDS-PAGE, including 7 major bands. All of the major bands are found in the internal pool and are seen to associate first with an endocytic compartment, and are then transferred to the lysosomes, reaching a peak in the lysosomes between 30 and 60 min. Material can then be transferred to the Golgi complex 1 to 3 h after labeling, and also, perhaps, to a post-Golgi compartment (which may be the GERL?) from which it recycles back to the cell surface. Characterization of the material associated with these various fractions by SDS-PAGE reveals that all the major bands are found in both the endocytic compartment and in lysosomes treated with NH₄Cl to impair degradation. In lysosomes from untreated cells, several bands are missing, and presumably degraded. Sorting is observed to occur at the level of internalization, during association of material with the endocytic compartment, and via specific degradation. No further sorting was observed after the material had been associated with the lysosom.al compartment.

Recommended Citation

Fishman, Jordan Barry, "Internalization, intracellular sorting, and recycling of cell surface sialoglycoproteins in HeLa cells. " PhD diss., University of Tennessee, 1984.
https://trace.tennessee.edu/utk_graddiss/12861