Isolation of a Golgi-rich fraction from rat liver

Leelavathi, D.E.; Estes, Larry W.; Feingold, David S.; Lombardi, Benito

doi:10.1016/0005-2736(70)90087-8

Cited by 236 publications

(98 citation statements)

References 23 publications

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“…As shown in Fig. 6B, we also detected such a protease activity in the detergent-solubilized Golgi fraction prepared from rat livers (27). These results suggest that the protease was localized mainly in the Golgi apparatus and its catalytic domain faces the luminal side.…”

supporting

confidence: 70%

Characterization of α2,6-Sialyltransferase Cleavage by Alzheimer's β-Secretase (BACE1)

Kitazume¹,

Tachida²,

Oka³

et al. 2003

Journal of Biological Chemistry

106

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BACE1 is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) at the ␤-secretase site, a critical step in the Alzheimer disease pathogenesis. We previously found that BACE1 also cleaved a membrane-bound sialyltransferase, ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I started at Glu 41 . Here we report that BACE1-Fc chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, between Leu 37 and Gln 38 , suggesting that an initial cleavage product by BACE1 was three amino acids longer than the secreted ST6Gal I. The three amino acids, Gln 38 -Ala 39 -Lys 40 , were found to be truncated by exopeptidase activity, which was detected in detergent extracts of Golgi-derived membrane fraction. These results suggest that ST6Gal I is cleaved initially between Leu 37 and Gln 38 by BACE1, and then the three-amino acid sequence at the NH 2 terminus is removed by exopeptidase(s) before secretion from the cells.

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supporting

confidence: 70%

Characterization of α2,6-Sialyltransferase Cleavage by Alzheimer's β-Secretase (BACE1)

Kitazume¹,

Tachida²,

Oka³

et al. 2003

Journal of Biological Chemistry

106

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“…The N-terminal sequence of the high-M, form, together with the observation that it is the major form observed immediately upon extraction of rat liver membranes, demonstrates that this Golgi-located enzyme is comprised of the near-intact translation product. This appears to be the first unambiguous evidence at the level of the protein sequence for purification of a membrane-bound glycosyltransferase; its isolation from rat liver, in particular, is pertinent as P1-4GalT has long been considered the marker enzyme for the Golgi apparatus based on subcellular frac-tionation studies performed with rat liver (MorrC et al, 1969(MorrC et al, , 1970Leelavathi et al, 1970).…”

Section: Discussionmentioning

confidence: 92%

“…The enzyme was initially discovered in particulate preparations from bovine and guinea pig mammary glands (Watkins and Hassid, 1961, 1 962). Later, /?I -4GalT was shown to be enriched about 30-100-fold in smooth membrane fractions from rat liver which were clearly identifiable morphologically as the conspicuous Golgi stack, using the appropriate exogenous acceptor, N-acetylglucosamine (MorrC et al, 1969(MorrC et al, , 1970Leelavathi et al, 1970). The protein was subsequently found to be concentrated in the transGolgi region (Roth and Berger, 1982).…”

mentioning

confidence: 99%

Proteins of the Golgi apparatus

Bendiak

Ward

Simpson

1993

European Journal of Biochemistry

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The Golgi marker enzyme, UDP‐galactose: N‐acetylglucosamine β1‐4galactosyltransferase (β1‐4GalT) was purified 44300‐fold in its intact, membrane‐bound form from rat liver membranes. The protein was isolated from detergent extracts as a high‐Mr form, having a Stokes radius approximating a globular protein of Mr 440000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an Mr near 51000, and does not have intermolecular disulfide crosslinks. N‐terminal sequencing of the enzyme demonstrated that it contains an N‐terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5′ end of the mRNA [Russo, R. N, Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324–3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N‐terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low‐Mr forms which were fully catalytically active and which, upon sequencing, were missing a 66‐amino‐acid stretch from the N‐terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with Mr near 50000. The N‐terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non‐protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

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“…Stacked Golgi (SG) fractions were prepared from rat livers as described (14) and immobilized on the solid support by mixing (3 hr at 4°C) with the ImAd in homogenization buffer adjusted to 0.25 M sucrose plus 0.5% bovine serum albumin. The separation of the bound from the unbound fraction was carried out using a magnet and the immobilized fraction was washed with the same buffer until minimal SG protein was present in the supernatant.…”

Section: Methodsmentioning

confidence: 99%

Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane proteins.

Salamero¹,

Sztul²,

Howell³

1990

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a cell-free assay that reproduces vesicular budding during exit from the Golgi complex. The starting preparation for the in vitro system was a rat liver stacked Golgi fraction immobilized on a magnetic solid support by means of an antibody against the cytoplasmic domain of the polymeric IgA receptor. Vesicular budding was ATP, cytosol, and temperature dependent and was inhibited by 1 mMN-ethylmaleimide. Budding was maximum within 10 min and originated preferentially from the trans-Golgi. Exocytic transport vesicles immunoisolated from the total budded population were enriched in the mature forms of secretory and membrane proteins destined to the basolateral plasma membrane and were depleted in lysosomal enzymes and galactosyltransferase activity. The finding that a major proportion (>70%) of newly synthesized, sialylated secretory and transmembrane proteins is contained in a single population of post-Golgi transport vesicles implies that, in a constitutively secreting cell, basolaterally destined proteins are sorted and packaged together into the same exocytic transport vesicles.The hepatocyte, a polarized epithelial cell, lacks storage granules or a known secretagogue, indicating that in this cell only a constitutive secretory pathway exists (1). This pathway delivers newly synthesized proteins to the blood and involves fusion of camer vesicles with the basolateral plasma membrane (PM). Transport of proteins into bile, mediated by fusion of vesicles with the apical PM, is by transcytosis rather than by direct traffic from the Golgi complex (2).Traffic of hepatocyte PM proteins seems to parallel that of secretory proteins. Bartles et al. (3) have presented evidence that PM proteins are initially transported to the basolateral PM, irrespective of their ultimate destination to either the basolateral or the apical PM. However, recent data suggest that sorting of apical and basolateral PM proteins can take place either intracellularly, at the trans-Golgi network (TGN), or at the basolateral PM depending on the protein and the cell type (4-6).Although it is likely that in constitutively secreting cells, PM and secretory proteins are transported from the Golgi to the cell surface in the same transport vesicles, there is currently no direct evidence for this in mammalian cells. Previous electron microscopic studies in hepatoma cells (7) indicated that a secretory protein (albumin) and a viral trans-membrane protein destined to the PM colocalize in structures within the Golgi region, some of which could represent exocytic transport vesicles.Several defined steps of biosynthetic protein traffic have been reconstituted in cell-free assays. Transport from the endoplasmic reticulum to the Golgi complex (8), between compartments of the Golgi complex (9), and from the Golgi complex to the plasma membrane (10, 11) has been investigated. In all of these studies the reporter molecules followed were viral proteins. We have designed a cell-free system that reconstitutes the formation and release of trans...

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Isolation of a Golgi-rich fraction from rat liver

Cited by 236 publications

References 23 publications

Characterization of α2,6-Sialyltransferase Cleavage by Alzheimer's β-Secretase (BACE1)

Characterization of α2,6-Sialyltransferase Cleavage by Alzheimer's β-Secretase (BACE1)

Proteins of the Golgi apparatus

Exocytic transport vesicles generated in vitro from the trans-Golgi network carry secretory and plasma membrane proteins.

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