High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

Abstract

The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0°C and warmed to 37C, the minimum pH observed was 6.1 ± 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24°C or 17°C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 ± 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.Iron delivery to vertebrate cells is primarily mediated by serum transferrin. Uptake of diferric transferrin has been shown to occur through receptor-mediated endocytosis in many different cell types (1-3), and both transferrin and receptor undergo subsequent acidification in the endocytic compartment (4, 5). Unlike most ligands, transferrin does not dissociate from its receptor upon acidification and therefore avoids lysosomal degradation by recycling with the receptor.Several lines of evidence point to the importance of a low endosomal pH in the effective delivery of iron: the increase in the rate of iron release from transfemn upon acidification in vitro (6); the inhibition of iron release caused by lysosomotropic amines known to raise intravesicular pH in vivo (1, 7-9); and the existence of Chinese hamster ovary (CHO) cell mutants defective in acidification, which cannot efficiently extract iron from transferrin (10-12 MATERIALS AND METHODS Preparation of Transferrin Conjugates. Human diferric transfemn was obtained from Miles Scientific (>98% pure). FITC and lissamine rhodamine sulfonyl chloride (LRSC) were obtained from Molecular Probes (Junction City, OR). Transferrin was conjugated with either FITC (FITC-Tf) or LRSC (LRSC-Tf) in 0.1 M sodium carbonate (pH 9.6). Free dye was removed using G-25 columns. The fluorochrome/ protein molar ratio was 4.3 for the FITC conjugate and 5.9 for the LRSC conjugate. These ratios were found to yield optimal specificity (90% or greater), and a signal/noise ratio (ratio of fluorescence from bound labeled transferrin to autofluorescence of unlabeled cells) of -3.0 for each conjugate. The mean number of transferrin binding sites per cell was determined to be 13,000 by using FITC-conjugated calibration b...

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.Differences in acidification may be important in determining the function of an endocytic compartment, because endocytic processes vary in their acidification requirements. Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0.…”

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.…”

“…Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0. Therefore, measuring the pH of a specific type of endosome may help elucidate the role that endocytic compartment has in a particular physiologic process.From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00 …”

“…From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00…”

“…From kinetic studies (12,29,32,40) it is not clear whether progressive acidification results from pH changes within an organelle or from passage among organelles that are regulated to different pH values. Several different types of endosomes have been described in CHO cells (34,39,41).…”