Differential association and distribution of acetyl- and butyrylcholinesterases within rat liver subcellular organelles

Rat liver cholinesterases were found to share properties and characteristics with those expressed in cholinergic tissues. The distribution and presence of different molecular forms of cholinesterases in different subcellular organelles of rat liver were studied. The rough and smooth endoplasmic reticulum and Golgi apparatus were enriched in the G4 molecular form of acetylcholinesterase (AChE) (relative to the G2 molecular form), while the inverse was found in the plasma membrane. The interaction of these molecular forms of AChE with the Golgi membrane was studied in detail. Approximately one-half of the G4 form was free within the lumen while the remainder was an intrinsic membrane protein; all the G2 molecular form was anchored to the membrane via phosphatidylinositol. Only the G1 and G2 molecular forms of butyrylcholinesterase (BuChE) were found in the above subcellular organelles; both molecular forms were soluble within the lumen of Golgi vesicles. These results indicate that rat liver expresses several molecular forms of AChE which have multiple interactions with membranes and that liver is unlikely to be the source of the G4 form of BuChE present in high concentration in the plasma.     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Expression of ceruloplasmin gene in various organs of the rat

The contribution of different rat organs to the synthesis of ceruloplasmin (Cp) was studied. Dot hybridization with the use of the Cp cDNA probe revealed Cp mRNA sequences in RNA preparations from liver, heart, kidney as well as from different divisions of brain, the concentration of Cp mRNA sequences being maximal in the liver. Polyribosomes isolated from these organs effectively synthesized Cp in a cell-free system derived from rabbit reticulocytes. After in vivo pulse labeling, the newly formed radioactive Cp was detected in the membrane fractions from all these organs. The newly formed Cp was concentrated within the membranes of the Golgi complex of various organs where it was revealed by different immunochemical techniques. Experiments with isolation of the liver from the systemic circulation showed that the liver is the only organ secreting Cp into the blood stream. It was suggested that mammalian tissues contain at least two molecular forms of Cp, i. e., circulatory and intracellular ones.     

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.     

The phaseolin vacuolar sorting signal promotes transient, strong membrane association and aggregation of the bean storage protein in transgenic tobacco

Vacuolar storage proteins of the 7S class are co-translationally introduced into the endoplasmic reticulum and reach storage vacuoles via the Golgi complex and dense vesicles. The signal for vacuolar sorting of one of these proteins, phaseolin of Phaseolus vulgaris, consists of a four-amino acid hydrophobic propeptide at the C-terminus. When this sequence is deleted, phaseolin is secreted instead of being sorted to vacuoles. It is shown here that in transgenic tobacco plants newly-synthesized phaseolin has unusual affinity to membranes and forms SDS-resistant aggregates, but mutated phaseolin polypeptides that are either secreted or defective in assembly do not have these characteristics. Association to membranes and aggregation are transient events: phaseolin accumulated in vacuoles is soluble in the absence of detergents and is not aggregated. Association to membranes starts before the phaseolin glycan acquires a complex structure and therefore before the protein reaches the medial or trans-cisternae of the Golgi complex. These results support the hypothesis of a relationship between aggregation and vacuolar sorting of phaseolin and indicate that sorting may start in early compartments of the secretory pathway.     

THE ANATOMY OF SECRETION IN THE FOLLICULAR CELLS OF THE THYROID GLAND : II. The Effect of Acute Thyrotrophic Hormone Stimulation on the Secretory Apparatus

This paper reports a study by phase contrast and electron microscopy of changes observed in the thyroid gland of the rat at 1, 2, 12, and 24 hours following an injection of thyrotrophic hormone. Examination by phase contrast microscopy reveals that follicular cells contain numerous colloid droplets 1 and 2 hours after injection. By 12 and 24 hours, the colloid droplets are no longer present, and individual follicles appear to be subdividing into smaller units. The droplets are assumed to contain newly synthesized colloid, and their development was studied by electron microscopy. During the period of active secretion, increase in number of the Golgi vesicles leads to enlargement of this organelle. At its periphery small colloid droplets appear to form from large Golgi vesicles. As they form, their content becomes more adielectronic, and fine dense particles less than 75 A in diameter appear in their matrix. Small- and medium-sized droplets lying in the apical region of the cell contain numerous dense particles scattered in their moderately adielectronic content. Large, mature droplets in the same region have a relatively dielectronic content resembling follicular colloid and no longer contain dense particles. The follicular cells appear to utilize apical pseudopodia to release the content of mature droplets into the follicular lumen. Other droplet-like inclusions occur in follicular cells, but they do not seem to be directly concerned with secretion.     

Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae. Precursor formation and processing in the yeast secretory pathway

A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged. Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells. We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Comparison of the complexed and free forms of rat liver arginyl-tRNA synthetase and origin of the free form

Arginyl-tRNA synthetase is found in multiple molecular weight forms in extracts from a variety of mammalian tissues. The rat liver enzyme can be isolated either as a component of the synthetase complex (Mr greater than 10(6) or as a free protein (Mr = 60,000). However, based on activity measurements after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the free form differs from its counterpart in the complex (Mr = 72,000). Both forms of arginyl-tRNA synthetase cross-react with an antibody directed against the complex, and both have similar catalytic properties. Thus, the two proteins have similar apparent Km values for arginine and ATP, the same pH optimum, are inhibited equally by elevated ionic strength and PPi, and they aminoacylate the same population of tRNA molecules. On the other hand, the free and complexed forms differ with respect to their apparent Km values for tRNA (free, 4 microM; complexed, 28 microM), their temperature sensitivity (complexed greater sensitivity), and their hydrophobicity (complexed more hydrophobic). Limited proteolysis of the synthetase complex with papain releases a low molecular weight form of arginyl-tRNA synthetase whose size, temperature sensitivity, and hydrophobicity are similar to that of the endogenous free form. Nevertheless, the usual 2:1 ratio of complexed-to-free form of rat liver arginyl-tRNA synthetase is not altered by a variety of homogenization or incubation conditions in the presence or absence of multiple protease inhibitors. In contrast to extracts of rat liver, rabbit liver extracts do not contain a free form of arginyl-tRNA synthetase. These results suggest that the complexed and free forms of arginyl-tRNA synthetase are probably the same gene product and that the free form in rat liver extracts is derived from the complexed form by a limited endogenous proteolysis that removes the portion of the protein required for anchoring it in the complex. The question of whether the free form is an artifact of isolation or whether it pre-exists in the cell is discussed.     

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions.Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.     

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.     

Animal DNA-dependent RNA polymerases. Purification and molecular structure of hen-oviduct and liver class-B RNA polymerases.

Hen ovidcut and liver class B RNA polymerases have been extensively purified and their molecular structure has been analysed. While only one enzyme B form (BIIb) was found in liver, three forms (BI, BIIa and BIIb) were resolved from oviduct. The molecular structures of the various class B RNA polymerase forms purified from hen oviduct and liver are identical to the corresponding forms previously purified from calf thymus and rat liver. At the present level of resolution the only difference lies in a slight difference in the charge of one subunit (SB2a) of enzyme form BIIa, when comparing the mammal and bird enzymes. It is unlikely that the absence of enzyme forms BI and BIIa in purified hen liver RNA polymerase B could be related to limited and specific proteolysis during the purification, since co-purification of oviduct and liver RNA polymerase B activities from a mixture of oviduct and liver nuclei does not affect the presence of either oviduct enzyme form BI or BIIa in the final purified mixture.     

Atrial natriuretic polypeptides (ANP): rat atria store high molecular weight precursor but secrete processed peptides of 25-35 amino acids

A radioimmunoassay was developed for rat atrial natriuretic polypeptides. Using the radioimmunoassay and gel filtration in reducing and dissociating conditions, extracts of rat atria were found to contain mainly a 15000-dalton immunoreactive material, probably corresponding to pronatriodilatin. However, when isolated beating atria were incubated in plasma-free conditions, the secreted immunoreactive material consisted almost exclusively of 2500-3500-dalton peptide(s). These results show that rat atrial cells secrete a low molecular weight natriuretic peptide which is highly active, but store the less active large molecular weight form(s).     

Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines

The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines.     

Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane

Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with [35S]Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.     

Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane contents of distinct subpopulations of coated vesicles determined by scanning transmission electron microscopy

In order to investigate the heterogeneity of clathrin-coated vesicles purified from rat liver, and to quantitate rigorously their membrane contents, we have analyzed scanning transmission electron micrographs of unstained coated vesicles before and after extraction with the non-ionic detergent Triton X-100, as well as of vesicles whose coats had been removed by dialysis against 10 mM or 100 mM Tris (pH 8.2). Their respective distributions of particle masses were thus determined and compared, in light of complementary biochemical quantitations of lipid and protein. Smaller coated particles, 25-45 MDa in mass and 60-80 nm in diameter, lose no mass when extracted with Triton, and disappear when their coats are dissociated. We conclude that they do not contain membrane vesicles, although they have dense, presumably proteinaceous, cores. They may represent particles generated during tissue homogenization or, possibly, a storage form of clathrin. The remaining 70% contain bona fide vesicles: these particles are 75-150 nm in diameter, and their average mass is about 80 MDa, of which 48 MDa is contributed by coat proteins, 10-12 MDa by phospholipid and cholesterol, and 20-22 MDa by vesicle-associated proteins. Their vesicles are of two types: smaller, denser, vesicles that contain substantial amounts of internalized material, and larger, less dense, vesicles that do not. The distinction between them may, in view of other findings, reflect a difference between coated vesicles derived respectively from the Golgi and the plasma membrane.     

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.     

Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance

Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.