Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways.

Abstract

Horseradish peroxidase (HRP), an enzyme internalized by fluid phase pinocytosis, has been used to study the process by which pinosome contents are delivered to lysosomes in Chinese hamster ovary cells. Pinosome contents were labeled by allowing cells to internalize HRP for 3-5 min . Following various chase times, cells were either processed for HRP and acid phosphatase (AcPase) cytochemistry or homogenized and fractionated in Percoll gradients . In Percoll gradients, pinosomes labeled by a 3-5-min HRP pulse behaved as a vesicle population more dense than plasma membrane and less dense than lysosomes. In pulse-chase experiments, internalized HRP was chased rapidly (3-6-min chase) to a density position intermediate between the "initial" pinocytic vesicle population and lysosomes. With longer chase periods, a progressive accumulation of HRP in more dense vesicles was observed . Correspondence between the HRP distribution and lysosomal marker distribution was reached after a^-1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRPpositive vesicles after pulse uptakes or a 3-min chase period was characterized by a peripheral rim of reaction product and was AcPase negative . After 10-120-min chase periods, the predominant class of HRP-positive vesicles was characterized by luminal deposits and HRP activity was frequently observed in multivesicular bodies . HRP-positive vesicles after a 10-or 30-min chase were AcPase-positive. No H RP activity was detected in Golgi apparatus . Together these observations indicate that progressive processing of vesicular components of the vacuolar apparatus occurs at both a prelysosomal and lysosomal stage .Prelysosomal endocytic compartments have recently been the subject of much interest in cell biology . These compartments, referred to variously as pinosomes, endosomes, intermediate vacuoles, or receptosomes (for reviews, see 6, 16, 31), have been described predominantly by electron microscopy and recently by cell fractionation . A major physiological function ofthese compartments appears to be ligand-receptor dissociation in response to vesicle acidification. In the present work, we have addressed the question of whether lysosomal as well as prelysosomal endocytic intermediates exist. Horseradish peroxidase (HRP'), an enzyme internalized by fluid phase pinocytosis in fibroblasts (1), has been chosen as the pinocytic marker because as a solute it should be included in all 'Abbreviations used in this paper: AcPase, acid phosphatase; CHO, Chinese hamster ovary; DAB, diaminobenzidine ; FC10, 10% fetal calf serum ; HRP, horseradish peroxidase ; aMEM, Eagle's minimum essential medium, alpha modification ; MIT, monoiodotyrosine . 108 pinocytic vesicles irrespective of their origin and because it can be readily assayed spectrophotometrically and localized cytochemically. A pulse-chase approach combined with cell fractionation and cytochemistry has been taken . The chief result of this work has been to demonstrate a rapid and progressive process...

The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

“…Mannose 6-phosphate receptors (M6PRs) 1 deliver newly synthesized acid hydrolases to the endocytic pathway and then return to the trans-Golgi network (TGN; Storrie et al, 1984;Brown et al, 1986;Duncan and Kornfeld, 1988;Kornfeld and Mellman, 1989). The route taken by M6PR-acid hydrolase complexes within the endocytic pathway has not yet been clearly defined but, from studies of the steady-state distribution of M6PR, it is generally believed that these receptors concentrate in a compartment on the endosometo-lysosome pathway, which has been called the prelysosome or late endosome (Griffiths et al, 1988(Griffiths et al, , 1990Kornfeld and Mellman, 1989).…”

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

“…Several different types of endosomes have been described in CHO cells (34,39,41). These include: (a) early, small vesicular and tubular endosomes often located near the cell surface, (b) large endosomes with diameters of 150-250 nm, and (c) recycling endosomes comprised of small vesicles and tubules located near the Golgi complex.…”

Fusion between endocytic vesicles in a cell-free system.

“…These kinetics make it unlikely that the bulk of the fusion events represent fusion of lysosomes with endosomes or other lysosomes, because endocytic markers in CHO cells only enter lysosomes after 10-15 min at 37°C (25). By default, therefore, endocytic compartments populated earlier in endocytosis are implicated in providing the bulk of the Biochemistry: Braell Proc.…”