The extent of exocytosis of pinocytic vesicle contents was studied in suspensioncultured Chinese hamster ovary (CHO) cells using horseradish peroxidase (HRP) as a pinocytic content marker. HRP was shown to be internalized via fluid-phase pinocytosis in CHO cells.After an HRP pulse of 2 .5-10 min a rapid decrease of 30-50% in cell-associated HRP activity was observed within 10-20 min at 37°C. During this time the loss of cell-associated HRP was accompanied by an equivalent increase in extracellular HRP. After this rapid exocytosis of HRP, the remaining peroxidase activity decreased with a t1/2 of 6-8 h, the known lysosomal half-life of HRP. In pulse-chase experiments HRP was chased into a nonexocytic compartment. Based on cell fractionation and electron microscopic experiments, this nonexocytic compartment was identified as a lysosome and the compartment from which exocytosis occurs as a pinosome . The occurrence of pinocytic content exocytosis in cultured fibroblasts suggests that exocytosis of pinocytic vesicle contents is a general phenomenon .

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...

Pinocytic vesicles (pinosomes) and lysosomes from suspension cultured, Chinese hamster ovary (CHO-S) cells have been resolved as two non-overlapping organelle populations by analytical centrifugation in Percoll gradients. Pinosomes were labeled with either horseradish peroxidase (HRP), a fluid phase content marker, or by radioiodination by pinocytosed lactoperoxidase (LPO). CHO-S cell lysosomes followed by three different marker enzymes and electron microscopy behaved as a single, dense organelle population. Pinosomes were partially resolved from plasma membrane, a less dense organelle population.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, 0. malhamensis shows a 10-to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. 0. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours.

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37°C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4°C. About 90% of the radioiodinated products pelleted at 100,000 x g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37°C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of -20 h. Upon warming cells to 37°C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10-to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of -90% of the radioiodinated membrane species initially present within the cell to the cell surface.The cell surface of mammalian cells is in a state of constant flux due to the continuous internalization of segments of the plasma membrane through the process of pinocytosis. Measurements of the rate of plasma membrane internalization by pinocytosis indicate that the equivalent of one cell surface can be internalized in less than 1 h without apparent changes in cell volume or surface area. For mouse fibroblasts, morphometric measurements (29) indicate that this cell type internalizes 0.5 cell surface equivalents per h. The soil amoeba Acanthamoeba has been estimated to internalize an amount of membrane equal to 10 to 40 cell surface equivalents per h (2). In contrast to the high rate of plasma membrane internalization, measurements made on the half-life of plasma membrane proteins by using enzymatic labeling with iodine (13), acetylation (20, 21), precursor incorporation (5), or studies of the turnover of specific membrane components (32) indicate that the degradation of most membrane proteins occurs slowly, with a tl/2 of 10 to 100 h.Plasma membrane proteins internalized through endocytosis can be transported to lysosomes over a time scale of 10 to 30 min. Radioiodinated plasma membrane proteins and marker enzymes present in latex phago-lysosomal membrane are rapidly lost (t1/2 of 1 to 2 h) from this compartment (13,37 261receptor for epidermal growth factor (4) or acetylcholine (7), are degraded. Very recent evidence indicates that phosphoryl mannose receptors in fibroblasts (26), a-macroglobulin receptors in macrophages (14), and asialoglycoprotein receptors in hepatocytes (28) are conserved. Experiments using anti-cell surface antibody as a tag for cell surface proteins (24) indicate that the antibody, and by inference cell surface proteins, can be shuttled without degradation between the cell surface and a lysosomal compartment. Radiolabeling data indicate pinocytic membrane proteins in the slime mold Dictyostelium discoideum (33), and phago-lysosome membrane proteins in macrophages (18,19)