The recycling of a secretory granule membrane protein

We have used N-hydroxysuccinimido-d-biotin as a reagent for labeling proteins exposed at the surface of cultured bovine adrenal chromaffin cells during Ba2+-stimulated secretion. A specific secretory granule membrane constituent, dopamine-beta-hydroxylase (DBH), has been investigated using immunoprecipitation followed by electrophoresis. Within 30 min of stimulation, exposed DBH had been cleared from the cell surface. Nevertheless, quantitation of labeled DBH using [125I] streptavidin suggested that it remained undegraded over a period of 24 h, a time during which secretory granule stores of catecholamines were being replenished. Subcellular fractionation of the cultured cells suggested that, after 3 or 4 h, the biotinylated DBH, which was still membrane-bound, was located in particulate material that also contained cytochrome b561, another major secretory granule membrane component.     

Identification and characterization of a melanocyte-specific novel 65-kDa peripheral membrane protein.

In order to study proteins of the melanosome, we developed a panel of antisera against various protein fractions of melanosomes from B16 melanoma cells. An antiserum raised against a Triton X-100 insoluble fraction of melanosomes recognized a 65-kDa protein in melanocytes from mice homozygous for the buff mutation, but not in their wild type counterparts. Further studies were conducted using a specific, second generation antiserum raised against the purified protein. The protein was also detected in melanocytes cultured from albino mice, but absent in cultured mouse cell lines not of melanocyte origin. Density gradient centrifugation of subcellular organelles and indirect immunofluorescent cell staining, indicated that the protein was associated with melanosomes and vesicles. The protein on intact organelles could be made soluble using sodium carbonate, and digested with proteases in the absence of detergent suggesting that it was a peripheral membrane protein localized on the cytosolic face of organelle membranes. Metabolic labelling of cells and N-glycosidase F digestion of cell extracts indicated that the protein was not N-glycosylated. Based on its intracellular localization and biochemical defects in the buff mouse, a potential role has been suggested for the 65-kDa protein in intracellular membrane trafficking.     

Purification and characterization of a 28-kDa major protein from ginseng root

A major protein was isolated from ginseng root (Panax ginseng C.A. Meyer) using a combination of ammonium sulfate fractionation, gel filtration chromatography, ion-exchange FPLC, and fast performance liquid chromatofocusing. Electrophoretic and gel permeation chromatographic studies revealed that the major protein, GMP, is composed of two subunits of approximately 28 kDa. During purification, it was found that the elution profiles of GMP from gel filtration chromatography were significantly different, depending on the ionic strength of buffers used. GMP in a buffer of low ionic strength was isolated as a complex with carbohydrate, which could be only dissociated at high ionic strength. Carbohydrate composition in GMP detected by gas chromatography varied, depending on the isolation method of the protein from ginseng roots. These results suggest that carbohydrates are bound non-covalently to GMP whose amino acid composition analysis showed high amounts of acidic amino acids.     

Trafficking of a secretory granule membrane protein is sensitive to copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.     

Identification of a 20-kDa protein with calcium uptake transport activity. Reconstitution in a membrane model

This paper presents results of experiments designed to further purify the membrane system involved in mitochondrial calcium transport. A partially purified extract, which transported calcium with a specific activity of 1194 nmol⁴⁵Ca²⁺/mg protein/5 min, was used to obtain mouse hyperimmune serum. This serum inhibited calcium uptake both in mitoplasts and in vesicles reconstituted with mitochondrial proteins containing cytochrome oxidase. Western blot analysis of the semipurified fraction showed that the serum recognized specifically two antigens of 75 and 20 kDa. Both antibodies were purified by elution from the nitrocellulose sheets and their inhibition capacity was analyzed. The antibody that recognized the 20-kDa protein produced a higher degree of inhibition than the other one.     

Interaction of alpha subunit of GTP-binding protein Go with a 20-kDa Triton-insoluble membrane protein in bovine brain.

Heterotrimeric Go bound to the membranes of bovine brain, but Go alpha remained bound to the membranes even after activation with GTP gamma S. Furthermore, Go alpha bound to a Triton X-100-insoluble fraction of the membranes in a saturable manner. However, the 37-kDa Go alpha eliminated by trypsin at the amino-terminus could not bind to the fraction. Using a blot overlay approach of the Triton-insoluble fraction, only a 20-kDa protein was identified that interacts with Go alpha. These results indicate that Go alpha binds to a 20-kDa Triton-insoluble protein in the bovine brain membranes.     

Characterization of the variability of a 75-kDa membrane protein in Mycoplasma hominis

The gene p75 encoding a 75-kDa surface-exposed membrane protein P75 was cloned and sequenced from Mycoplasma hominis type strain PG21T. To investigate the intraspecies variability, sequences were obtained from an additional two isolates 7488 and 183, and the three sequences were compared. The nucleotide and amino acid differences were not confined to specific regions of the gene/protein, but when comparing the three sequences, differences were present as single site substitutions or small insertions or deletions of nucleotides/amino acids. The intraspecies variability was further investigated by restriction enzyme analysis with two restriction enzymes (Alul and MboII) of PCR products amplified from p75 from 28 M. hominis isolates. On the basis of band patterns produced by the two restriction enzymes, the isolates could be divided into five and six groups. These groups neither matched categories of the M. hominis vaa gene nor the M. hominis p120 gene classes, indicating that the three genes vary by different mechanisms and possibly indicating horizontal gene transfer. Federation of European Microbiological Societies.     

Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae.

Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag.     

Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal medullary chromaffin granule membranes by immunoblotting

An antiserum which recognizes high molecular mass enkephalin-containing proteins was used to compare proenkephalin intermediates in both the soluble and membrane components of bovine adrenal chromaffin granules by immunoblotting. While a range of molecular mass forms were identified in the soluble lysate the major form in the membranes corresponded to a 27-kDa enkephalin-containing protein. Enzymic digestion of bands of 27-kDa material and quantitation of the enkephalin released showed that 22% of this material was membrane-associated. High concentrations of chaotropic agents were required to extract this material from the membranes. Association of hormone and neuropeptide precursors with membrane components may be important for targeting of precursors to secretory granules or correct processing.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

Phosphorylation of a 51-kDa envelope membrane polypeptide involved in protein translocation into chloroplasts

In this report we demonstrate that a 51-kDa outer-envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membrane-impermeable agent pyridoxal 5'-phosphate, which has been shown to react with a component of the protein-import apparatus. Phosphorylation of envelope membrane polypeptides using [gamma-32P]ATP in the presence of pyridoxal 5'-phosphate resulted in an increased incorporation of 32P radiolabel into a 51-kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [gamma-32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer-envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51. Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 microM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP-dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein-import apparatus or the protein receptor itself.     

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2'',5''-phosphodiester bond (RNA branch) with the 5'' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.     

Leishmania donovani: characterization of a 38-kDa membrane protein that cross-reacts with the mammalian G-protein transducin

We investigated the presence in Leishmania donovani promastigotes of proteins with homology to the G-proteins known to mediate signal transduction in other organisms. [alpha 32P]GTP binding experiments revealed the presence in the promastigote membrane of GTP-binding sites with high affinity and specificity. Experiments with antisera directed against mammalian G-proteins showed that the promastigotes possess a 38-kDa protein (p38) which strongly reacts with an antiserum directed against a decapeptide containing the C-terminal sequence of transducin, the G-protein that mediates visual signal transduction. The interaction of p38 with the antiserum is specifically blocked by the decapeptide antigen. p38 is enriched in plasma membranes and is absent in cytosol and in a mitochondria-enriched fraction. p38 was also detected in two other Leishmania species, L. mexicana and L. major. The migration of p38 upon sucrose gradient centrifugation of detergent extract of L. donovani membranes corresponded to Mr of approximately 70,000, indicating that p38 is part of an oligomeric structure. The findings suggest that p38 may be a component of a transmembrane signal transduction system in Leishmania.     

Purification of Chlamydomonas 28-kDa ubiquitinated protein and its identification as ubiquitinated histone H2B.

One of the most predominantly ubiquitinated protein species in Chlamydomonas, of which the apparent molecular mass in SDS-PAGE was 28 kDa, was found to exist abundantly in nuclei. The 28-kDa ubiquitinated protein was purified to homogeneity from the isolated nuclei of Chlamydomonas, and its partial amino acid sequence was determined. The N-terminal peptide sequence was identical with that of ubiquitin. Sequences homologous to those Chlamydomonas ubiquitin [corrected] and wheat histone H2B, and paired sequences of both of them were found in arginylendopeptidase-digested or protease V8-digested polypeptide fragments of the 28-kDa ubiquitinated protein. Based on these results, it was concluded that Chlamydomonas 28-kDa ubiquitinated protein is monoubiquitinated histone H2B.     

Isolation and characterization of a 40-kDa cyclophilin-related protein.

Major and minor isoforms of cyclophilin (CyP-18), a 17.8-kDa protein with peptidyl-prolyl cis-trans isomerase activity, comprise the primary intracellular binding proteins for cyclosporin A. Additional CyP-like proteins with approximate molecular masses of 22 (CyP-22) and 40 kDa (CyP-40) have been recovered from the soluble fraction of calf brain along with CyP-18 by adsorption onto a cyclosporin A affinity column and elution with cyclosporin A. Based on a limited number of peptide sequences from CyP-22, it appears that we have isolated from tissue CyPB, a protein whose sequence was deduced previously from cloned cDNA. The 40-kDa protein was separated from CyP-18 and CyP-22 on a molecular sieving column. Isoelectric focusing of CyP-40 yielded two bands at pI 5.3 and 5.5, in contrast to the basic pI values of CyP-18. Some tryptic peptides from CyP-40 were found to be highly homologous but not identical to bovine CyP-18; others were not significantly homologous to CyP-18 or any other protein in the data base. Unlike the major and minor isoforms of Cyp-18, monospecific polyclonal anti-CyP-18 antibodies did not cross-react with CyP-22 and CyP-40. Likewise, anti-CyP-40 serum minimally cross-reacts with CyP-18 and CyP-22. Cyp-40 possesses peptidyl-prolyl cis-trans isomerase activity which is less sensitive to inhibition by cyclosporin A (IC50 = 300 nM) than is CyP-18 (IC50 = 20 nM).     

Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

Photoactivatable (-²³P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the³²P-labeled proteins were found to be 62-and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the³²P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphryldolichol synthase.     

The major zymogen granule membrane protein GP-2 in the rat pancreas is not involved in granule formation.

The major membrane protein of zymogen granules in the rat pancreas is a glycoprotein of 78 kDa (GP-2), which is inserted into the membrane via a glycosyl-phosphatidylinositol (GPI) anchor. GP-2 occurs in both, a membrane-attached and a soluble form. Due to its specific luminal orientation and its quantitative contribution to the zymogen granule membrane, GP-2 has been postulated to play an important role in sorting of digestive enzymes into the granule and in the formation of the granule as a storage organelle. We have tested this hypothesis in the rat pancreas under three different functional conditions, where both the rates of enzyme/isoenzyme synthesis change drastically, and new zymogen granules form at a high rate: a) during prolonged hormonal stimulation of the adult rat pancreas, b) during the differentiation of AR4-2J cells induced by dexamethasone in vitro, and c) during embryonic development and early postnatal life, when gene expression is modulated due to the differentiation program. Both, GP-2 mRNA levels and the rate of GP-2 biosynthesis were quantitated and compared to the immunohistochemical localization of this protein in tissue sections. Under all three functional conditions, significant changes could be demonstrated at the level of digestive enzyme gene expression, but no concomitant modulation of GP-2 expression was observed. GP-2 mRNA is absent from the embryonic pancreas and for the first time is expressed after birth with a significant increase during the period of weaning. Furthermore, GP-2 mRNA and protein levels are not modulated by hormonal stimulation, either in the adult pancreas or in AR4-2J cells in culture. Therefore, we conclude that GP-2, in spite of its quantitative contribution to the zymogen granule membrane, is not involved in enzyme protein sorting or granule formation. Alternative functions for GP-2 are discussed.     

Purification of a 175-kDa membrane protein, its localization in smooth and cardiac muscles. Interaction with cytoskeletal protein - vinculin

A new 175-kDa membrane protein was isolated from chicken gizzard smooth muscle. Antibodies to 175-kDa protein were used for localization of this protein in smooth and cardiac muscles. In both types of muscle 175-kDa protein was localized near plasma membrane. 175-kDa protein was able to interact specifically with vinculin immobilized on polysterene surface. It is suggested that this 175-kDa protein may be involved in physical connection between microfilaments and cell membrane.