An essential yeast gene encoding a homolog of ubiquitin-activating enzyme.

Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.

Kinetic mechanism of phosphate/phosphate and phosphate/OH- antiports catalyzed by reconstituted phosphate carrier from beef heart mitochondria.

In an optimized reconstituted system, the basic kinetic properties of the phosphate carrier from bovine heart mitochondria, e.g. the influence of membrane potential, pH, and proton gradient, were investigated for the two physiological modes of transport (Pi-/Pi- antiport and electroneutral, unidirectional phosphate transport). On the basis of these data, which closely resemble the function known from mitochondria, the reaction mechanism of the phosphate carrier was determined using bireactant initial velocity studies in both transport modes. Translocation occurred according to a simultaneous (sequential) mechanism, involving a ternary complex in transport catalysis. This mechanism indicates that the phosphate carrier falls into the same functional family as most other mitochondrial carriers. A detailed analysis of the different effects of pH on transport substrates and carrier protein in both possible transport modes, in combination with the identity of the kinetic mechanism in both modes, provides evidence that the unidirectional phosphate transport is catalyzed by Pi-/OH- antiport rather than by Pi-/H+ symport. We furthermore observed noncompetitive inhibition of phosphate transport by other anions. The consequences of this result with respect to a functional model of the carrier protein are discussed.

Functional properties of the reconstituted phosphate carrier from bovine heart mitochondria: evidence for asymmetric orientation and characterization of three different transport modes.

The phosphate carrier from bovine heart mitochondria was reconstituted into liposomes by the removal of detergent using hydrophobic ion-exchange columns. Reversible blocking of the carrier function during chromatographic steps was possible by the application of the inhibitor p-(chloromercuri)benzenesulfonate at low temperature. Thus, both forward and backward exchange experiments for kinetic characterization of Pi/Pi-antiport as well as the Pi/H(+)-symport could be performed. The maximum rate of Pi/Pi-antiport was 90 mumol min-1 (mg protein)-1. Only one single half-saturation constant (Km) for phosphate was observed at each side of the membrane under antiport conditions, 1.8 mM at the external and 9.4 mM at the internal side. By comparing the Km values at both sides of the membrane with the values found in intact mitochondria, a right-side-out orientation of the reconstituted phosphate carrier was concluded. Furthermore, the influence of various sulfhydryl reagents on the carrier was investigated. After modification with HgCl2, the phosphate carrier reveals a third (nonphysiological) unidirectional transport mode. This was characterized by a significantly reduced substrate specificity. In view of similar observations with several other mitochondrial carriers, these results again indicate that the phosphate carrier is a member of the postulated functional family of mitochondrial carrier proteins.

Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria. Structure/function relationship involving one lysine and two cysteine residues.

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 microM) 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.

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

Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400 microM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS, IC50 300 microM) could specifically be suppressed by the substrates aspartate and glutamate; a 50% substrate protection was observed at half-saturation of the external binding site. The same held true for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, IC50 500 microM) and diethyl pyrocarbonate (DEPC, IC50 20 microM), two reagents known to modify carboxylic or histidinyl side-chains, respectively. EDC, however, turned out to catalyze an acylation of the active site lysine by activating carboxyls that had to be present in the incubation medium. This special mechanism, which was proven by protein labelling using EDC/[14C]succinate, necessitates a lysine side-chain of high reactivity and low pK, since the modification had to occur at pH less than or equal to 6.5, i.e. not too far from the pK of the carboxyl to be activated. All reagents applied, additionally including 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, IC50 10 microM), were effective at this pH. Competition experiments revealed interaction of EDC, PLP, SITS and probably DIDS at the same active site lysine. For DEPC a lysine modification could not be ruled out. Yet, a model comprising a histidine juxtaposed to the lysine seems to be appropriate.