A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough endoplasmic reticulum

Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.     

A conformationally altered precursor to the lysosomal enzyme alpha-mannosidase accumulates in the endoplasmic reticulum in a mutant strain of Dictyostelium discoideum

The mutant strain of Dictyostelium discoideum, HMW-437, contains a mutation in the structural gene coding for the lysosomal enzyme alpha-mannosidase. Unlike the wild type strain, Ax3, this strain fails to proteolytically process or secrete the 140,000-dalton alpha-mannosidase precursor. The level of sulfate incorporation into the mutant precursor was significantly lower when compared to the wild type precursor. In addition, the mutant precursor was entirely sensitive to endoglycosidase H. Subcellular fractionation of HMW-437 membranes indicated that the majority of the alpha-mannosidase precursor sedimented in a region of the gradient corresponding to the rough endoplasmic reticulum. This accumulation within the rough endoplasmic reticulum did not appear to result from gross conformational changes which lead to aggregation. Trypsin digestion of radioactively labeled Ax3 and HMW-437 precursors demonstrated that there were differences in susceptibility to protease cleavage between the wild type and mutant alpha-mannosidase precursor molecules, suggesting that a minor conformational change could contribute to the accumulation of the mutant precursor inside the endoplasmic reticulum.     

Sulfated N-linked oligosaccharides affect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum

Although previous studies have indicated that N-linked oligosaccharides on lysosomal enzymes in Dictyostelium discoideum are extensively phosphorylated and sulfated, the role of these modifications in the sorting and function of these enzymes remains to be determined. We have used radiolabel pulse-chase, subcellular fractionation, and immunofluorescence microscopy to analyze the transport, processing, secretion, and sorting of two lysosomal enzymes in a mutant, HL244, which is almost completely defective in sulfation. [3H]Mannose-labeled N-linked oligosaccharides were released from immunoprecipitated alpha-mannosidase and beta-glucosidase of HL244 by digestion with peptide: N-glycosidase. The size, Man9-10GlcNAc2, and processing of the neutral species were similar to that found in the wild type, but the anionic oligosaccharides were less charged than those from the wild-type enzymes. All of the negative charges on the oligosaccharides for HL244 were due to the presence of 1, 2, or 3 phosphodiesters and not to sulfate esters. The rate of proteolytic processing of precursor forms of alpha-mannosidase and beta-glucosidase to mature forms in HL244 was identical to wild type. The precursor polypeptides in the mutant and the wild type were membrane associated until being processed to mature forms; therefore, sulfated sugars are not essential for this association. Furthermore, the rate of transport of alpha-mannosidase and beta-glucosidase from the endoplasmic reticulum to the Golgi complex was normal in the mutant as determined by the rate at which the newly synthesized proteins became resistant to the enzyme, endo-beta-N-acetylglucosaminidase H. There was no increase in the percentage of newly synthesized mutant precursors which escaped sorting and were secreted, and the intracellularly retained lysosomal enzymes were properly localized to lysosomes as determined by fractionation of cell organelles on Percoll gradients and immunofluorescence microscopy. However, the mutant secreted lysosomally localized mature forms of the enzymes at 2-fold lower rates than wild-type cells during both growth and during starvation conditions that stimulate secretion. Furthermore, the mutant was more resistant to the effects of chloroquine treatment which results in the missorting and oversecretion of lysosomal enzymes. Together, these results suggest that sulfation of N-linked oligosaccharides is not essential for the transport, processing, or sorting of lysosomal enzymes in D. discoideum, but these modified oligosaccharides may function in the secretion of mature forms of the enzymes from lysosomes.     

A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha- mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild- type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N- linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.     

Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.     

Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates

We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta-glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.     

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe- AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.     

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum

We have examined the relationship of N-linked oligosaccharide structures to the proper targeting and proteolytic processing of two lysosomal enzymes, alpha-mannosidase and beta-glucosidase, in the slime mold Dictyostelium discoideum. Two different mutant strains, HL241 and HL243, each synthesize the same nonglucosylated, truncated, lipid-linked oligosaccharide precursor, Man6GlcNAc2. [3H]Mannose-labeled N-linked oligosaccharides were studied following their release from immunoprecipitated alpha-mannosidase and beta-glucosidase by digestion with peptide:N-glycosidase F. The oligosaccharides from both mutants resembled each other, but they were smaller and contained fewer anionic groups than those from the wild-type. The oligosaccharides from the mutants strains were reduced in sulfate and Man-6-P content, and all Man-6-P was in the form of acid-stable phosphodiesters. Pulse-chase radiolabeling experiments using [35S] methionine indicated that the precursor forms of both enzymes were smaller than wild-type, and that this difference was due solely to differences in N-linked oligosaccharides. The precursor forms of the enzymes were not over-secreted, but appeared to be proteolytically processed into mature forms at approximately 50% the rate of wild-type. This is mainly due to their prolonged retention in the rough endoplasmic reticulum, but, ultimately, both enzymes were properly targeted to lysosomes. These studies indicate that a reduction in the amount of sulfation, phosphorylation or size of the N-linked oligosaccharides in these mutants is not critical for the proteolytic processing and targeting of the lysosomal enzymes, but that these changes may influence their rate of exit from the rough endoplasmic reticulum.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.     

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.     

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters

Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study I have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake, and that each tetrameric, holoenzyme molecule has sufficient carbohydrate for an average of 10 Man8GlcNAc2 oligosaccharides. None of the oligosaccharides or glycopeptides from the lysosomal enzymes bind to an immobilized PMR, but those with 2 PDE show slight interaction. Competition of 125I-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Furthermore, the uptake of a lysosomal enzyme isolated from a mutant strain (modA), which produces oligosaccharides with only 1 but not 2 PDE, is about 10-fold less than the uptake of wild-type enzyme which has predominantly 2 PDE. Complete denaturation of 125I-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes. The conformation of the protein may be important in orienting the oligosaccharides in a favorable position for binding to the PMR.     

Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways

Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.     

A deletion in the golgi alpha-mannosidase II gene of Caenorhabditis elegans results in unexpected non-wild-type N-glycan structures

The processing of N-linked oligosaccharides by alpha-mannosidases in the endoplasmic reticulum and Golgi is a process conserved in plants and animals. After the transfer of a GlcNAc residue to Asn-bound Man(5)GlcNAc(2) by N-acetylglucosaminyltransferase I, an alpha-mannosidase (EC 3.2.1.114) removes one alpha1,3-linked and one alpha1,6-linked mannose residue. In this study, we have identified the relevant alpha-mannosidase II gene (aman-2; F58H1.1) from Caenorhabditis elegans and have detected its activity in both native and recombinant forms. For comparative studies, the two other cDNAs encoding class II mannosidases aman-1 (F55D10.1) and aman-3 (F48C1.1) were cloned; the corresponding enzymes are, respectively, a putative lysosomal alpha-mannosidase and a Co(II)-activated alpha-mannosidase. The analysis of the N-glycan structures of an aman-2 mutant strain demonstrates that the absence of alpha-mannosidase II activity results in a shift to structures not seen in wild-type worms (e.g. N-glycans with the composition Hex(5-7)HexNAc(2-3)Fuc(2)Me) and an accumulation of hybrid oligosaccharides. Paucimannosidic glycans are almost absent from aman-2 worms, indicative also of a general lack of alpha-mannosidase III activity. We hypothesize that there is a tremendous flexibility in the glycosylation pathway of C. elegans that does not impinge, under standard laboratory conditions, on the viability of worms with glycotypes very unlike the wild-type pattern.     

Identification of mannose 6-phosphate receptors in rabbit alveolar macrophages

Mannose 6-phosphate is an important recognition site involved in transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes. The current study is the first demonstration of functional mannose phosphate receptors in macrophages. The receptor appears to be similar in many respects to that expressed in fibroblasts. Binding at 4 degrees C of a mannose-6-P-containing ligand, alpha-mannosidase from Dictyostelium discoideum, was specific and saturable (KD = 1.6 nM). In the presence of permeabilizing agents (saponin and digitonin), macrophage mannose-6-P receptors gave a distribution of 15-20% on the surface and 80-85% inside. Uptake studies gave a Kuptake value of 4.9 nM. Mannose-6-P, Hansenula holstii phosphomannan, and fructose 1-phosphate were effective inhibitors of alpha-mannosidase uptake. Inhibitors of mannose uptake, such as beta-glucuronidase, mannose-bovine serum albumin, fucose-bovine serum albumin, or mannan had no effect on alpha-mannosidase uptake. Likewise, an inhibitor (fucoidin) of the macrophage receptor which recognizes negatively charged proteins did not inhibit alpha-mannosidase uptake. Uptake was linear over 90 min and inhibited by chloroquine, suggesting that surface receptors recycle. These data demonstrate that macrophages contain receptors which specifically recognize mannose-6-P units and are distinct from the well characterized mannose receptors. The finding that the mannose-6-P receptors play a role at the surface, together with the fact that most of the receptors are intracellular (similar to the mannose receptor) suggests that both carbohydrate receptors play a regulatory role at the surface and intracellularly in transport of lysosomal enzymes.