Studies on the internalization and intracellular transport of horseradish peroxidase in Chinese hamster ovary cells

Abstract

Soluble horseradish peroxidase (HRP) is internalized by Chinese hamster ovary cells, a cell line of fibroblastic origin (Adams et al., 1982). We have confirmed this result by showing no inhibition of uptake in the presence of divalent cation chelators (EGTA Mg or EDTA), excess (19 mg/ml) yeast mannan (an inhibitor of uptake through a mannose/N-acetylglucosamine receptor) or using periodate treated HRP. Periodate treatment destroys the ring structure of sugars on HRP which have hydroxyl groups on adjacent ring carbons, eliminating sugar mediated uptake of HRP. Once internalized, HRP is found in endocytic vesicles which by HRP-cytochemical staining, show deposits which rim the luminal face of vesicle membrane. Once HRP is in lysosomes, cytochemical deposits are luminal. To test if HRP is actually associated with vesicle membrane, a hypotonic lysis assay was used. Postnuclear supernatants (PNS) from cells pulse labeled with HRP were lysed and the percent of HRP sedimenting with a high speed membrane fraction was used as a measure of membrane association. After a pulse, >60% of the total HRP internalized was pelletable. Hypotonic lysis of a PNS at different pH and temperature showed no significant difference in "pelletability" from 4℃ to 37℃ at neutral pH and only a slight decrease in "pelletability" with increased temperature (4℃ to 37℃) at pH M.6. Binding of HRP in a membrane preparation was pH and temperature stable. Uptake of native HRP in the presence of yeast mannan (19 mg/ml) or using periodate treated HRP also had little effect on "pelletability", suggesting the absence of sugar specific binding in endocytic vesicles. Using the hypotonic lysis assay of a PNS after different chase times, HRP dissociation from membrane was observed over a 30 minute chase period. Internalized HRP in the presence of yeast mannan (19 mg/ml), intravesicular pH elevators HEPES (40 mM) or monensin (10 μM), or substances which should deplete cellular ATP NaF/KCN (2 mM /1 mM), showed no inhibition of dissociation kinetics. A chase at 17℃ inhibited dissociation of HRP over the entire 30 minute period. This HRP binding site(s) appears unique to endocytic vesicles.

A minimum of four steps in transport have been identified based on their sensitivity to inhibitors. HRP transport, identified by Percoll density gradient fractionation, was inhibited at 17°C and was sensitive to pH elevators (NH₄Cl, monensin, HEPES) and ATP depletion (NaF/KCN). Inhibition of transport appeared to be independent of HRP dissociation except at early temperature sensitive step(s). These results suggest that transport inhibition may be due to an effect on a) inhibition of membrane dissociation (early step(s)) and alteration of membrane fluidity (later steps) by reduced temperature and b) transmembrane events by pH elevators and ATP depletion.