Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites

Hudson, Robert Tod; Draper, Rockford K.

doi:10.1091/mbc.8.10.1901

Cited by 33 publications

(37 citation statements)

References 35 publications

(39 reference statements)

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“…Aminoglycoside antibiotics such as neomycin have been previously shown to effectively precipitate COPI complexes (42,43). As a potential way to corroborate our results showing ␣-chain sequence-dependent COPI association, we tested the capacity of neomycin to co-precipitate various Tac reporter molecules expected to show varying degrees of COPI association.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

Cauvi

Tian

Loehneysen

et al. 2006

Journal of Biological Chemistry

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The human high affinity IgE receptor (Fc⑀RI) is a central component of the allergic response and is expressed as either a trimeric ␣␥2 or tetrameric ␣␤␥2 complex. It has been previously described that the cytoplasmic domain (CD) of the ␣-chain carries a dilysine motif at positions ؊3/؊7 from the C terminus that functions in intracellular retention prior to assembly with other Fc⑀RI subunits. In this report we have further explored the role of the ؊3/؊7 dilysine signal in controlling steady-state ␣-chain transport by mutational analysis and found little surface expression of a ؊3/؊7 dialanine ␣-chain mutant but significant Golgi localization. We compared the transport properties of a series of ␣-chain cytoplasmic domain truncation mutants and observed that truncation mutants lacking 23 or more C-terminal residues showed a dramatic increase in steady-state transport suggesting a role for the membrane-proximal CD sequence in ␣-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys The high affinity IgE receptor (Fc⑀RI) 2 is a multisubunit complex comprised of either a tetrameric ␣␤␥2 complex or an alternative trimeric ␣␥2 isoform (1, 2). Aggregation of the tetrameric receptor on mast cells and basophils occurs upon cross-linking of receptor-bound IgE with a multivalent antigen that initiates Fc⑀RI-dependent signaling, culminating in cellular degranulation and the ensuing clinical symptoms of hypersensitivity. Expression of the trimeric Fc⑀RI isoform occurs on a number of cells, including monocytes, epidermal Langerhans cells, and dendritic cells, and a role for the ␣␥2 receptor in antigen presentation has now been established (3). The Fc⑀RI ␣-chain is exclusively involved in IgE binding, whereas the associated ␥-and ␤-chains function in signaling by the presence of intracellular tyrosine activation motifs. Assembly with the ␤-chain also functions to amplify Fc⑀RI cell-surface expression relative to the ␣␥2 isoform (4, 5) and represents a fundamental mechanism for the regulation of Fc⑀RI cell-surface expression. It has long been known that binding of IgE to cell-surface Fc⑀RI leads to enhanced receptor expression (6), an effect that has now been amply demonstrated for both receptor isoforms (6 -9) and is thought to involve receptor-ligand surface stabilization and possibly increased export of ␣-chain from an intracellular pool (10, 11). Additional mechanisms that regulate Fc⑀RI expression have also been identified, including the effects of interleukin-4 in mast cells (7, 12) and transforming growth factor-␤1 in both dendritic cells (13) and mast cells (14). Because the sensitivity of the allergic response is critically linked to Fc⑀RI surface expression (15) that in turn is dependent on Fc⑀RI subunit transport characteristics (16) and assembly (17), it remains an important goal to fully elucidate the Fc⑀RI structural features that regulate these processes to thereby provide a better understanding of the molecular origins of the allergic response.The underlying factors th...

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Section: Resultsmentioning

confidence: 99%

“…Neomycin Precipitation of COPI Complexes-COPI complexes were precipitated from transfectant lysates essentially as previously described (42,43). Briefly, HEK293 transfectants were suspended in lysis buffer (0.1% Triton X-100, 25 mM HEPES, 50 mM KCl, 2.5 mM Mg(OAc) 2 , pH 7.4) and solubilized by cavitation using a 25-gauge syringe needle.…”

Section: Construct Namementioning

confidence: 99%

Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

Cauvi

Tian

Loehneysen

et al. 2006

Journal of Biological Chemistry

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“…Two different binding sites within coatomer have recently been suggested for dilysine motifs on the basis of the observation that interaction of coatomer with certain aminoglycoside antibiotics, which would mimic a pair of dilysine residues, leads to precipitation of the complex (37). This, however, would imply more than one binding site for amino groupcontaining structures within coatomer and is in contrast to the results of this study, which provide strong evidence for a single binding site for both dilysine retrieval motifs and the cytoplasmic domain of p23.…”

Section: Discussionmentioning

confidence: 99%

A single binding site for dilysine retrieval motifs and p23 within the γ subunit of coatomer

Harter

Wieland

1998

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

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Coatomer, the major component of the coat of COPI transport vesicles, binds both to the dilysine motif of resident membrane proteins of the endoplasmic reticulum and to the cytoplasmic domain of p23, a major type I membrane protein of COPI vesicles. Using a photocrosslinking approach, we find that under native conditions a peptide analogous to the cytoplasmic domain of p23 interacts with coatomer exclusively through its ␥ subunit and shares its binding site with a KKXX retrieval motif. However, upon dissociation of coatomer, interaction with various subunits, including an ␣-, ␤-, -COP subcomplex, of the photoreactive peptide is observed. We suggest that, under physiological conditions, interaction of coatomer with both endoplasmic reticulum retrieval motifs and the cytoplasmic domain of p23 is mediated by ␥-COP.

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“…Coatomer from mammalian cells is known to interact with a dilysine (KKXX) motif present in the cytoplasmic domain of membrane proteins known to travel from the Golgi to the ER (Letourneur et al, 1994; for review, see Lowe and Kreis, 1998). Neomycin contains paired amino groups at three separate locations in the molecule, and it has been suggested by Hudson and Draper (1997) that coatomer has a binding site that accommodates two amino groups such as those present in neomycin. As a result, neomycin effectively cross-links coatomer into large, sedimentable aggregates.…”

Section: Cytosolic Atsec21p and Atsec23p Belong To Different Protein mentioning

confidence: 99%

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

et al. 1999

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Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cellshas not yet been demonstrated, although the GTPbinding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membranebound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-densitygradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

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Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites

Cited by 33 publications

References 35 publications

Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

A single binding site for dilysine retrieval motifs and p23 within the γ subunit of coatomer

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

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