Purification and characterization of lysosomes from Chinese hamster ovary cells

Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with [35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts.     

Fibroblasts maintain a complete endocytic pathway in the presence of lysosomotropic amines

We have investigated the effects of the lysosomotropic amines, ammonium chloride and chloroquine, on the delivery of fluid-phase pinocytic tracers to lysosomes in Chinese hamster ovary (CHO) cells. In preliminary experiments, 15 mM ammonium chloride and 0.1 mM chloroquine were found to be sufficient to give maximal protection of endocytosed material from digestion in a lysosome. In the presence of either amine at these concentrations, the generation time of CHO cells was depressed by less than 30% even though selective depletion of lysosomal hydrolases was observed. For cells treated with either amine for 1 or 6 days the amount of horseradish peroxidase (HRP) internalized in a 1-h pulse was approximately 50-70% of that of control. By cell fractionation, cells treated with amine for 2 or 6 days were found to accumulate fluorescein-dextran or HRP in lysosomes. HRP accumulation in lysosomes in amine-treated cells was also observed by electron microscopy. Little exocytosis of lysosomal HRP into the media was observed under any condition. We conclude that in long-term amine-treated CHO cells endocytic vesicle traffic is maintained.     

Retention of pinocytized solute by CHO cell lysosomes correlates with molecular weight

We compared the exocytosis by Chinese hamster ovary (CHO) cells of a set of fluid-phase pinocytic tracers. The tracers were horseradish peroxidase (HRP), a glycoprotein of approximately 40 kDa, lucifer yellow (LuY), a 457 dalton, membrane-impermeant fluorescent dye, and glucose polymers ranging from sucrose through higher molecular weight, fluorescein isothiocyanate (FITC) dextrans. After a long term uptake (16-20 h), each of these tracers was localized to lysosomes. Exocytosis of the majority of the small molecule tracers, LuY and [14C] sucrose, was observed over a period of a few to several h. There was no significant exocytosis of 42 kDa FITC dextran or HRP during an 18-20 h chase, while lower molecular weight dextrans were exocytosed. After co-accumulation of LuY and HRP in lysosomes, only the low molecular weight marker was exocytosed. These observations suggest retention of endocytized solutes within lysosomes is dependent on molecular size and may be limited by the rate of diffusion of molecules into shuttle vesicles.     

Biochemical evidence for an endocytically inactive population of lysosomes

The peroxidase dependent, diaminobenzidine (DAB) density shift procedure was applied to the characterization of lysosomes from Chinese hamster ovary (CHO) cells. Peroxidase activity was localized in lysosomes by a 15-18 h internalization period. After treatment with DAB, the distribution of peroxidase activity in Percoll gradients was shifted, as a population, to a higher density. A bimodal distribution which included a low density population was observed for the native lysosomal enzyme beta-hexosaminidase after DAB treatment. A second lysosomal enzyme, alpha-fucosidase, was strongly inhibited by DAB treatment with the residual activity corresponding in distribution to the light beta-hexosaminidase population. The occurrence of a low density lysosomal population after the DAB procedure suggests the existence of an endocytically inactive lysosomal population in fibroblasts. Probable physiological candidates for such a population are discussed.     

Characterization of pinocytic vesicles from CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation

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.     

Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways

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 approximately 1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRP- positive 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 HRP 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.     

Exocytosis of pinocytic contents by Chinese hamster ovary cells

The extent of exocytosis of pinocytic vesicle contents was studied in suspension-cultured 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 degrees 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.     

IκB Is a Substrate for a Selective Pathway of Lysosomal Proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor κ B (IκB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IκB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IκB is directly transported into isolated lysosomes in a process that requires binding of IκB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IκB uptake by lysosomes. Ubiquitination and phosphorylation of IκB are not required for its targeting to lysosomes. The lysosomal degradation of IκB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IκB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IκB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IκB is increased. There are both short- and long-lived pools of IκB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IκB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IκB degradation in lysosomes. This selective pathway of lysosomal degradation of IκB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor κ B. In addition, the response of nuclear factor κ B to several stimuli increases when this lysosomal pathway of proteolysis is activated.     

A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation

We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Effect of acidotropic amines on the accumulation of newly synthesized membrane and luminal proteins in Chinese-hamster ovary (CHO) cell lysosomes.

Lysosomes were isolated by sequential gradient centrifugation [Madden, Wirt & Storrie (1987) Arch. Biochem. Biophys. 257, 27-38] from control or acidotropic-amine-treated Chinese-hamster ovary (CHO) cells. By marker-enzyme analysis, the preparation from chloroquine or NH4Cl-treated cells was about 25-fold enriched for lysosomes compared with the postnuclear supernatant and contained little or no marker activities for the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, mitochondria, cytosol and peroxisomes. The yield of amine-treated lysosomes was about 60% relative to the postnuclear supernatant. Electron microscopy and cytochemistry demonstrated that the amine-treated preparation was highly purified. Cytochemical analyses after a short-term pulse of horseradish peroxidase revealed that endosomal contamination of the lysosomal preparation was less than 1%. Lysosomal polypeptides were biosynthetically labelled with [35S]methionine and identified by SDS/polyacrylamide-gel electrophoresis. As expected, the bulk accumulation of luminal proteins into lysosomes was decreased. The bulk accumulation of membrane proteins was increased by acidotropic amine treatment. There were also several qualitative differences in each lysosomal compartment, with new species observed and other species absent. These data suggest that a low pH is not necessary for the normal accumulation of the bulk of membrane proteins in lysosomes and that membrane trafficking from Golgi apparatus to lysosomes occurs at a high rate in acidotropic-amine-treated CHO cells.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.     

Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles

We investigated the effects of reduced temperature, the pH elevators NH4Cl, monensin, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from "early" to "late" (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17 degrees C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long-term HRP uptakes at 17 degrees C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from "very early" endosomes occurred at 17 degrees C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the "early" endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that "early" steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.     

Chinese hamster ovary cell mutants defective in myo-inositol transport

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.     

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.     

Endocytic membrane traffic with respect to phagosomes in macrophages infected with non-pathogenic bacteria: phagosomal membrane acquires the same composition as lysosomal membrane

A morphometric analysis was made to study membrane traffic in bone marrow-derived macrophages, containing phagosomes with partially degraded Bacillus subtilis. Cell surface glycoproteins, labeled with radioactive galactose by terminal glycosylation, provided a covalent autoradiographic membrane marker. Membrane compartments were characterized in terms of cytochemical staining for horseradish peroxidase taken up by receptor-mediated endocytosis. The area, composition, and exchange rates of endocytic membrane compartments were measured as in a previous analysis for non-infected macrophages, devoid of phagosomes. In direct comparison with this earlier study, the present data allowed an assessment of the involvement of phagosomes in the interactions between endocytic membrane compartments. The presence of phagosomes led to a 30% reduction of lysosomal membrane area. The rate at which cell surface-derived label flowed into the lysosomal membrane pool was reduced by the same fractional amount. This suggested a linear relationship between flow rate and membrane area. The initial flow rate of label into phagosomes was higher than expected, based on their membrane area being only about 60% that of lysosomes. This rate could only be measured during the early phase of the experiments when phagosomes were younger, therefore displaying a fast exchange rate, reminiscent of the endosome compartment. However, steady-state conditions, at late times, strongly suggested that phagosomes with degraded contents finally acquire membrane of lysosomal origin. First, the composition of phagosome membrane became the same as that of lysosomes, remaining unchanged as compared to non-infected cells. Second, the membrane area of phagosomes amounted to the loss of lysosomal membrane area in infected cells.