Probing the active site of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed acylation of a functional lysine residue

Dierks, Thomas; Stappen, Reiner; Salentin, Angelika; Krämer, Reinhard

doi:10.1016/0005-2736(92)90052-n

Cited by 19 publications

(16 citation statements)

References 45 publications

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“…The gating mechanism obviously is triggered by specific recognition of substrates at the active site, which no longer is the case in the uniport state. Consistently, we recently characterized a single essential lysine residue within the exofacial binding site of the AGC, the modification of which led to complete inhibition of both transport functions, antiport as well as uniport [16].…”

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confidence: 63%

“…4A, only mersalyl, Nbs, and HgCl, shown), thus suggesting reaction with the same cysteine. In contrast, carrier pretreatment with diethyl pyrocarbonate [O(COzC2H&], which was shown also to react at the binding site, probably with a histidine residue [16], did not affect labeling with Nbd-C1 (Fig. 4B).…”

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 95%

“…Since the reaction of a variety of SH-and lysine reagents with the AGC has already previously been characterized [S, 14,16,39], it was important to examine the mutual interaction of these reagents and Nbd-CI with the carrier protein. Being available in labeled form, [14C]Nbd-C1 could furthermore be applied to monitor the accessibility of the active-site cysteine after modification of the AGC with other reagents.…”

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 99%

“…4B). Modification of the active-site lysine [16], on the other hand, clearly interfered with the incorporation of ['4C]Nbd-C1. Interestingly, the degree of protection against Nbd-C1 labeling was correlated with the size of the lysine- modifying reagents applied.…”

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 99%

“…Biophys. Acta 1103, [13][14][15][16][17][18][19][20][21][22][23][24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively.…”

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confidence: 99%

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Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria

Stappen

Dierks

Krämer

1992

European Journal of Biochemistry

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Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (Nbd‐Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or glutamate, 50% substrate protection being observed at half‐saturation of the external binding site. The bifunctional reagent 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS) also modified the same cysteine and, in addition, an active‐site lysine identified previously [Dierks, T., Stappen, R., Salentin, A. & Krämer, R. (1992) Biochim. Biophys. Acta 1103, 13–24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively. By using a variety of reagents a further cysteine [Cys(b)] and probably a histidine residue could be discriminated from Cys(a) and the lysine. The applied reagents were classified according to functional and structural criteria. Class A reagents, like Nbd‐Cl, modified the active‐site Cys(a) thereby inhibiting the antiport function. Class B reagents, like HgCl2, reacted with both Cys(a) and Cys(b) leading to a conversion of the carrier from antiport to uniport function [Dierks, T., Salentin, A., Heberger, C. & Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268–280]. DIDS at relatively high concentration (60 μM) also acted as a uniport inducer. Class C reagents finally, like pyridoxal phosphate or diethyl pyrocarbonate, modified the active‐site lysine or histidine, respectively, and blocked antiport and uniport activity. By testing the accessibility of the mentioned residues to the various reagents, when applied in different order, topological relationships could be elaborated indicating the location of these amino acids with respect to the exofacial active site of the carrier protein.

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confidence: 63%

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 95%

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 99%

Section: Reaction Of Nbd-ci With the Aspartatelglutamate Carriermentioning

confidence: 99%

mentioning

confidence: 99%

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Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria

Stappen

Dierks

Krämer

1992

European Journal of Biochemistry

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An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

Mayinger¹,

Meyer²

1993

The EMBO Journal

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The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 × 10(−5) M. ATP transport into ER vesicles was specifically inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3′‐O‐(4‐benzoyl)benzoyl‐ATP (Bz2‐ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2‐ATP blocked protein translocation in vitro measured co‐ as well as post‐translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS‐treated membranes. These results demonstrate that translocation requires a DIDS and Bz2‐ATP‐sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.

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Mitochondrial Transport Processes

Kaplan

1996

Molecular Biology of Membrane Transport Disorders

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Probing the active site of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed acylation of a functional lysine residue

Cited by 19 publications

References 45 publications

Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria

Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

Mitochondrial Transport Processes

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