Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes.

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.     

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

β-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of β-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

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.     

Secretion of alpha-L-fucosidase by human embryonal skin fibroblasts

The amount of alpha-L-fucosidase secreted by normal human fibroblasts was higher in the medium containing 10% bovine serum than in the medium containing 0.1% bovine serum. Glycosidase secretion was twice higher at the advanced than at the initial stage of subcultivation. Extracellular activity of alpha-L-fucosidase from 3 different fibroblast strains differed insignificantly in the medium containing 0.1% bovine serum, while intracellular activity of the enzyme in these strains was altogether different. The results suggest that the lysosomal glycosidase secretion is determined by the level of cellular endocytosis.     

Rod/cone dysplasia in Irish setters. Presence of an altered rhodopsin.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.     

Effect of temperature on extracellular accumulation of beta-D-N-acetylglucosaminidase in I-cell disease fibroblast cultures.

The activities of most lysosomal enzymes are elevated in the culture fluid of skin fibroblasts derived from patients with I-cell disease with a corresponding reduction in the intracellular activities when the cells are cultured at 37°C. When I-cell fibroblast cultures are incubated at 27°C for 8–24 hr, the β-D-N-acetylglucosaminidase (EC 3.2.1.30) activity accumulated in the culture fluid is reduced to approximately 25% of the activity in 37°C control cultures without a corresponding change in intracellular activity. No significant effect of temperature is observed on the intra- and extracellular distribution of β-D-N-acetylglucosaminidase in non-I-cell fibroblast cultures. These findings suggest the existence of two lysosomal enzyme pools in I-cell fibroblasts, one of which is temperature-dependent and destined for excretion while the other remains intracellular and appears to be unaffected by temperature.     

Effect of the co-existence of galactosyl and phosphomannosyl residues on β-hexosaminidase on the processing and transport of the enzyme in mucolipidosis I fibroblasts

Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compare the properties of intra- and extracellular β-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete β-hexosaminidase when maintained in serum-free medium. Using the specifity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted β-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that β-hexosaminidase molecules contained phosphomanosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of β-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.     

Electrophoretic abnormalities of lysosomal enzymes in mucolipidosis fibroblast lines.

Electrophoretic properties of eight lysosomal hydrolases and 36 nonlysosomal enzymes were investigated in cultured fibroblasts from children with the inherited storage disease mucolipidosis II (ML II); fibroblasts from a child with a related disorder, mucolipidosis III (ML III); and two obligate heterozygous cell lines from parents of a ML II child. Cell homogenates of ML II fibroblast lines showed altered mobilities for lysosomal beta-hexosaminidase, acid phosphatase2, and alpha-mannosidase and deficient activity for the esterase-A4 and lysosomal alpha-mannosidase-B electrophoretic phenotypes. Altered mobility was also detected for the nonlysosomal enzyme adenosine deaminase-d. Deficient activities of other lysosomal enzymes were observed as previously reported. In a single ML III fibroblast line, only beta-hexosaminidase showed an abnormal electrophoretic pattern suggesting a difference between these cells and ML II fibroblasts. Thirty-five nonlysosomal enzymes associated with other cellular organelles and metabolic pathways were electrophoretically normal in all mucolipidosis cell lines. Heterozygous ML II cells showed normal expression for all enzymes. Two major patterns of altered lysosomal enzymes and adenosine deaminase were demonstrated in ML II cell lines, suggesting that at least two genetic forms of this disorder may exist. Neuraminidase treatment of ML II homogenates converted altered forms of acid phosphatase2 and adenosine deaminase-d and in two ML II lines, recovered the previously undetected lysosomal alpha-mannosidase band. These results are consistent with the mucolipidosis defect(s) being associated with abnormal post-translatinal processing of multiple lysosomal enzymes and adenosine deaminase-d.     

Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts.

1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Inhibition by cyanate of the processing of lysosomal enzymes

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.     

Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.

The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.     

Intercellular exchange of lysosomal hydrolases between mutant human fibroblasts and other cell types

Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes.     

Distribution of acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.     

The effect of monensin on beta-hexosaminidase transport in normal and I-cell fibroblasts.

The carboxylic ionophore, monensin, blocks the migration of glycoprotein-containing vesicles from the Golgi region to the plasma membrane in fibroblasts resulting in an accumulation of secretory products in the Golgi cisternae. Treatment of cultured I-cell fibroblasts with monensin (0.5 muM) decreased the abnormal excretion of beta-hexosaminidase to 40% of untreated cultures within 15 min. A corresponding intracellular accumulation of the enzyme to greater than 200% of untreated cultured by 24 h was also observed. A small intracellular accumulation and slightly enhanced excretion of beta-hexosaminidase occurred in treated normal fibroblasts cultures. The intra- and extra-cellular distribution of newly synthesized beta-hexosaminidase in both monensin-treated normal and I-cell fibroblasts were electrophoretically indistinguishable from the four bands characteristic of I-cell intracellular beta-hexosaminidase. The excreted enzyme from both cultures was found to be a low- or no-uptake form. This form of beta-hexosaminidase may have been excreted from a secondary route preceding the site of the monensin effect. The similar findings in monensin-treated normal and I-cell cultures suggest that the subcellular site of the biochemical defect in I-cell disease is at a location after the site of the monensin effect i.e. late in the Golgi region or at a post-Golgi-region location.     

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.     

Tilorone acts as a lysosomotropic agent in fibroblasts

Tilorone, an amphiphilic cationic compound with antiviral activity perturbed the lysosomal system. In cultured fibroblasts tilorone induced storage of sulfated glycosaminoglycans, enhanced secretion of precursor forms of lysosomal enzymes, inhibited intracellular proteolytic maturation of lysosomal enzymes, and inhibited receptor-mediated endocytosis of lysosomal enzymes. In isolated lysosomes tilorone was found to increase pH and to abolish the ATP-dependent acidification. These effects suggest that tilorone acts like a weak base that accumulates in acid compartments of the cells, raises the pH therein and interferes with lysosomal catabolic activity and with receptor-mediated transport of lysosomal enzymes.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.