Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins.

Abstract

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase ([3H]gaIBSA-HRP), injected 10 min before sacrifice . These organelles were recovered at low density (1 .11-1 .13 g/ml) in sucrose gradients (Quintart, J ., P. J. Courtoy, J. N . Limet, and P. Baudhuin, 1983, Eur. J. Biochem ., 131 :105-112) . Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H 202 and equilibration in a second sucrose gradient, gaIBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J ., J . Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB-stained structures represented -85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities . These minor activities could reflect either contamination or true enzyme association to the ligandcontaining structures . Considering the latter hypothesis, at most 1 .0% of alkaline phosphodiesterase I and 2 .6% of 5'-nucleotidase (markers for plasma membrane), 3 .6% of N-acetyl-ß-glucosaminidase (lysosomes), and 6 .0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand-containing organelles . After DAB cytochemistry on liver fixed 10 min after gaIBSA-HRP injection, ligand-containing structures accounted for 0 .78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2 /Cm 3, compared with 6,700 Cm2/Cm 3 for the pericellular membrane . Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex .Rapidly after interiorization, numerous ligands taken up by receptor-mediated endocytosis (7,31,44) are found associated with structures equilibrating at low density (15, 21-23, 26, 30, 35, 40, 41, 48). Electron microscopy shows that ligands are concentrated in clathrin-coated pits or vesicles, and rapidly transferred into electron-lucent, smooth surfaced organelles (35,45,47

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

“…Other structures remained essentially at the initial low density, as indicated by the distributions of marker enzymes for the plasma membrane, lysosomes, and the Golgi complex (not shown) . The density distributions of these marker enzymes are presented in detail in the companion paper (24). Since the density shift induced by ga1BSA-HRP ("active" ligand) caused a concomitant density shift of ga1BSA ("passive" ligand), but not of other components, this experiment demonstrated that both derivatives were localized within the 874 THE IOURNAL OE CELL BIOLOGY " VOLUME 9S, 1984 same organelles.…”

“…The specificity ofthe density shift to ligand-HRP containing structures was demonstrated by in vitro mixing experiments, a criterion already used by Leskes et al (19). In addition, only a minor portion of the protein shifted after cytochemistry, and the distribution of several marker enzymes present in the initial preparation was largely unaffected (24).…”

“…The reaction was started by the addition of 50~1 of 6% HZ Oz, prepared extemporaneously by dilution of a 30% H20z stock solution in sucrose-imidazole, and performed for 30 min at 25°C, in the dark, under gentle agitation . The H,Oz concentration in the incubation medium ( l I mM) was measured by the TiOSOe z See Materials and Methods of the companion paper (24) for an explanation of the fraction nomenclature . colorimetric assay (4).…”

“…Particles without HRP will remain at their initial density. Application of this strategy to the purification of the low density ligand-containing structures is reported in the companion paper (24).…”

“…In comparison, the DAB-induced density shift takes advantage of a highly sensitive, exogenous enzyme, that can be introduced into specific organelles along the endocytic pathway by fluid-phase or receptor-mediated endocytosis (7,23,27,29,33). In addition, this procedure is simple, fairly reproducible, and proved useful to analyze and to purify ligand-HRP-containing organelles (24). This work has already been presented in abstract form (5) .…”