The sequential transfer of internalized, cell surface sialoglycoconjugates through the lysosomes and Golgi complex in HeLa cells.
Surface sialoglycoproteins of HeLa cells were labeled by NaB[3H]4 reduction after oxidation with NaIO4, yielding seven major radioactive bands as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When labeled cells are reincubated in growth medium, all of these major classes of glycoproteins are internalized and all but one (105 kDa) are recycled, i.e. subsequently reappear on the surface. The surface-labeling patterns over time remain qualitatively similar, but changes in relative specific activity of the bands suggest some preferential degradation of individual glycoproteins. Analytical fractionation at various time points after labeling suggests that the surface molecules pass through the lysosomal compartment and subsequently accumulate in the Golgi and Golgi-related compartments before returning to the surface. Inhibition of lysosomal function with chloroquine or NH4Cl prevents the accumulation and subsequent recycling. The pathway is confirmed with preparative fractionation into surface membrane, prelysosomal, lysosomal, Golgi, and Golgi-related compartments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrates a degree of preferential handling of the glycoproteins on this pathway, e.g. the 180-kDa band is relatively reduced at the endocytic/prelysosomal stage and the 105-kDa band appears to be degraded in its first passage through the lysosomes. The other bands recycle 10-20 times before being degraded.
D000643:Ammonium Chloride / Q000494:pharmacology; D001894:Borohydrides / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D012795:Sialoglycoproteins / Q000378:metabolism*; D014316:Tritium
D001894:Borohydrides; D012795:Sialoglycoproteins; D000643:Ammonium Chloride; D014316:Tritium; D002738:Chloroquine