Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

The physiology of Clostridium sporogenes NCIB 8053 growing in defined media

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy.     

The ATP-dependent interaction of eukaryotic initiation factors with mRNA

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor

Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.