Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.

Tan, Adrienne; Bolscher, Jan G. M.; Feltkamp, Constance A.; Ploegh, Hidde L.

doi:10.1083/jcb.116.6.1357

Cited by 32 publications

(21 citation statements)

References 55 publications

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“…While the disassembly of BFA-treated Golgi is inhibited by the nonhydrolyzable guanine nucleotide analog GTP'yS in permeabilized cells (20,53), this has not been demonstrated in vivo. To determine the .extent to which GTPases control BFA-induced Golgi disassembly and reassembly, we introduced GTP'tS into the cytoplasm of REF-52 cells via microinjection.…”

Section: Both the Dispersal Of The Goigi With Bfa And Its Reassembly mentioning

confidence: 96%

“…In this study we explore the possibility that rabia protein is not only actively involved in anterograde vesicular traffic, but also is critical for maintaining the structural integrity of the Golgi apparatus. This work follows the observations that BFA may inhibit the guanine nucleotide exchange protein that is critical for recruitment and/or maintenance of ARF on Golgi membranes (17, 28), and that GTP~,S (which targets all guanine nucleotide binding proteins) and A1F4-(believed to target heterotrimeric G proteins [31]) interfere with Golgi apparatus disassembly after exposure to BFA (18,34,53).…”

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confidence: 99%

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A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

Wilson

Nuoffer

Meinkoth

et al. 1994

The Journal of Cell Biology

122

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Abstract. The Golgi apparatus is a dynamic organdie whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for mbla, a GTPase previously associated with ER to Golgi and intra-Golgi transport, 'in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rabla protein was mieroinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rabla(S25N), a constitutively GDP-botmd form

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Section: Both the Dispersal Of The Goigi With Bfa And Its Reassembly mentioning

confidence: 96%

mentioning

confidence: 99%

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

Wilson

Nuoffer

Meinkoth

et al. 1994

The Journal of Cell Biology

122

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“…Preparation of Subcellular Fractions from Rat Liver or HT29 Cells and Cell Permeabilization-Cells were permeabilized with SL-O according to the procedure of Tan et al (26), using 1.3 units/ml SL-O. Efficiency of permeabilization (Ͼ90%) was checked in every experiment by counting propidium iodide-labeled (permeabilized) and total cells.…”

Section: Methodsmentioning

confidence: 99%

Dihydroceramide Biology

Kok¹,

Nikolova‐Karakashian²,

Klappe³

et al. 1997

Journal of Biological Chemistry

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This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-DH-Cer (C 6 -NBD-DHCer) was incubated with HT29, NRK, BHK, or HL-60 cells, it was efficiently converted to dihydrosphingomyelin and dihydroglucosylceramide, and a number of other sphingolipids, with the nature of the products depending on the cell line. In addition, complex sphingolipids were formed that contained a desaturated (sphingosine) backbone, indicating that DH-Cer (and/or its metabolites) were substrates for the desaturase(s) that introduce the 4,5-trans double bond. Based on the kinetics and inhibitor studies, double bond addition did not appear to occur with the complex sphingolipids directly, but rather, during turnover and resynthesis. The conversion of C 6 -NBD-DH-Cer to more complex sphingolipids was highly stereoselective for the natural D,erythro isomer of C 6 -NBD-DH-Cer. Interestingly, the stereochemistry of the sphingoid base backbone also affected the localization of fluorescent sphingolipids: the D,erythro species appeared in the Golgi apparatus, whereas other stereo-isomers accumulated in the endoplasmic reticulum. In addition to C 6 -NBD-Cer and C 6 -NBD-DH-Cer, C 6 -NBD-4-D-hydroxy-DH-Cer gave rise to formation of complex sphingolipids and localized at the Golgi apparatus. These studies indicate that dihydroceramide is used as the initial backbone of complex (glyco)sphingolipids, perhaps to avoid build up of ceramide as an intermediate since this is such a potent bioactive compound. The stereoselectivity in transport and metabolism suggests that trafficking of ceramide is protein-directed rather than simply a consequence of vesicular membrane flow.

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“…Optimal SLO and digitonin concentrations were determined to yield permeabilization of more than 95% of the cells, as judged by uptake of trypan blue and release of lactate dehydrogenase. This 135-kDa cytosolic enzyme was estimated by its activity measured spectrophotometrically as a decrease in A 340 due to NADH oxidation (reaction mixture: 50 mM sodium phosphate buffer (pH 7.4), 1 mM NADH, 1% Triton X-100, 5 mM sodium pyruvate (modified from Tan et al (34)). Permeabilized cells were resuspended in RPMI medium and chased in vitro by incubation at 37°C in a humid CO 2 -air incubator.…”

Section: Methodsmentioning

confidence: 99%

Degradation of Distinct Assembly Forms of Immunoglobulin M Occurs in Multiple Sites in Permeabilized B Cells

Winitz

Shachar

Elkabetz

et al. 1996

Journal of Biological Chemistry

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Protein degradation is essential for quality control which retains and eliminates abnormal, unfolded, or partially assembled subunits of oligomeric proteins. The localization of this nonlysosomal pre-Golgi degradation to the endoplasmic reticulum (ER) has been mostly deduced from kinetic studies and carbohydrate analyses, while direct evidence for degradation within the ER has been provided by in vitro reconstitution of this process. In this article, we took advantage of the transport incompetence of permeabilized cells to directly demonstrate that the selective degradation of secretory IgM (sIgM) in B lymphocytes is transport-dependent. We show that, upon permeabilization of the plasma membrane with either streptolysin O or digitonin, sIgM is not degraded unless transport is allowed. Nevertheless, upon complete reduction of interchain disulfide bonds with thiols, the free heavy chains are degraded by a transport-independent quality control mechanism within the ER. This latter degradation is nonselective to the secretory heavy chain s, and the membrane heavy chain m, which is normally displayed on the surface of the B cell, is also eliminated. Moreover, the degradation of free s is no longer restricted to B lymphocytes, and it takes place also in the ER of plasma cells which normally secrete polymers of sIgM. Conversely, when assembled with the light chain, the degradation is selective to sIgM, is restricted to B lymphocytes, and is a transport-dependent post-ER event.Protein degradation plays a key role in a myriad of regulatory processes (1). A quality control mechanism is responsible for retention and elimination of abnormal, unfolded, or partially assembled subunits of oligomeric proteins (2-4). Metabolically controlled turnover is reported for various proteins, such as HMG-CoA 1 reductase (5, 6), apolipoprotein B (apoB) (7-10), and ornithine decarboxylase (11). The quality control mechanism operates within the endoplasmic reticulum (ER) and is probably assisted by the variety of ER chaperones (12, 13). Degradation in a nonlysosomal pre-Golgi compartment is reported for numerous proteins, including the ␣ and ␤ subunits of the T cell receptor (14 -16), the H2a subunit of the asialoglycoprotein receptor (17-19), a truncated form of ribophorin (20), the PiZ variant of ␣ 1 -antitrypsin (21), unassembled immunoglobulin light chain (22), and two mutated yeast vacuolar proteins (23), as well as HMG-CoA reductase (5, 6) and apoB (7-10). Localization of these processes to the ER has been directly demonstrated only for the yeast proteins (23). In most cases, however, it has been deduced from temperature and ATP requirements, kinetic studies, structure of carbohydrate moieties, and insensitivity to drugs that block export of proteins from the ER. Secretory IgM (sIgM) is an oligomeric protein that is rapidly degraded in B lymphocytes but is efficiently secreted from plasma cells (24,25). We have shown that, in the 38C B lymphocytes, sIgM degradation is also nonlysosomal and occurs prior to the trans-Golgi (26, 27). More...

show abstract

Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.

Cited by 32 publications

References 55 publications

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

Dihydroceramide Biology

Degradation of Distinct Assembly Forms of Immunoglobulin M Occurs in Multiple Sites in Permeabilized B Cells

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