Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Subramaniam, V.N.; Yusoff, A. R. Bin Mohd; Wong, Siew Heng; Lim, Gaik Bee; Chew, Chee-Meng; Hong, Wanjin

doi:10.1016/s0021-9258(19)49799-8

Cited by 26 publications

(3 citation statements)

References 47 publications

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“…Membranes were prepared as described (Subramaniam et al, 1992), with some minor modifications. Livers obtained from Sprague-Dawley rats which had been fasted overnight were rinsed once in icecold PBS and then in homogenization buffer (0.25 M sucrose, 25 mM Hepes, pH 7.4, 5 mM MgCl2, 1 mM PMSF).…”

Section: Preparation Of Membrane Fractionsmentioning

confidence: 99%

Preferential association of syntaxin 8 with the early endosome

Subramaniam

Loh

Horstmann

et al. 2000

Journal of Cell Science

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Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ((alpha)-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.

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Section: Preparation Of Membrane Fractionsmentioning

confidence: 99%

Preferential association of syntaxin 8 with the early endosome

Subramaniam

Loh

Horstmann

et al. 2000

Journal of Cell Science

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“…Fractions were treated with various reagents for the removal of sequentially different types of proteins (Subramaniam et al, 1992). The first treatment was with 10 mM EGTA in 25 mm Tris-HCl, 5 mM MgCl2 buffer, pH 7.3, which solubilizes peripheral proteins.…”

Section: Purification Of Parotid Gland Kinasementioning

confidence: 99%

Purification of Kinase Activity from Primate Parotid Glands

Lamkin

Lindhe

2001

J Dent Res

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Salivary secretions contain phosphoproteins that contain phosphorylation sites composed of serine residues in acidic environments. The hypothesis of this study is that a protein kinase responsible for phosphorylating these proteins is similar to kinases that phosphorylate proteins in other glandular secretions. Homogenates and subfractions from macaque parotid glands were able to phosphorylate synthetic peptide substrates containing each of the phosphorylation sites in acidic proline-rich proteins, statherin, and histatin 1. Activity was purified from Golgi membranes to greater than 220-fold by extraction with Triton X-100 and affinity chromatography with the use of immobilized ATP. The enzyme preferred substrates containing serine residues in a specific acidic environment, particularly those containing the Ser-Xaa-acidic sequence, preferred ATP over GTP, and was sensitive to high concentrations of heparin. These characteristics are similar to those reported for Golgi casein kinase, which phosphorylates casein in vivo. Based on these observations, the parotid gland kinase may be related to other Golgi-localized serine kinases.

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“…The transfection reagent DOTAP {N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulfate} and antiimmunoglobulin G antibodies coupled to fluorescein or rhodamine were from Boehringer Mannheim. Monoclonal antibodies (clone 9E10) to c-myc were from Oncogene Science while polyclonal antisera to p23 (KDEL receptor) and ,B-1,4galactosyltransferase (GT) have been previously described (22,25). The human breast carcinoma cell line BT-20 was from the American Type Culture Collection.…”

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confidence: 99%

Transmembrane Topology of the Mammalian KDEL Receptor

Singh

Tang

Wong

et al. 1993

Molecular and Cellular Biology

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The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.

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Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Cited by 26 publications

References 47 publications

Preferential association of syntaxin 8 with the early endosome

Preferential association of syntaxin 8 with the early endosome

Purification of Kinase Activity from Primate Parotid Glands

Transmembrane Topology of the Mammalian KDEL Receptor

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