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.

All three genes (AACl.AAC2and AAC3) encoding the mitochondrial ADP/ATP translocator. were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of .Gcchorornyces cermisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROXl factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using ZacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROXl factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROXl factor.The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADPIATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX%, HEM13 and CYC7 that are negatively regulated by oxygen and heme.In Saccharomyces cerevisiae the mitochondria1 ADP/ATP translocator exists as three isozymes that are encoded by distinct nuclear genes, AACI, AAC2, and AAC3 [l-31. The reason for the existence of multiple genes encoding a protein functioning in ADP/ATP translocation across mitochondrial membrane is not apparent. Gene-disruption experiments have shown that the product of AAC2 gene is the major protein involved in ADP/ATP exchange across the mitochondrial membrane and that it is essential for oxidative phosphorylation [2-41. The disruption of the AACl or AAC3 gene did not yield a distinct phenotype, but the combination of these mutations with other nuclear mutations resulted in phenotypes indicating a vacuolar role for the AACZ product and a role in the anaerobic metabolism for the AAC3 product [3, It has been shown that AAC genes are differentially regulated to express separate isofoms under different physiologi-51. and that a functional ADP/ATP translocator is essential for anaerobic growth of yeast cells [9].The expression of several other yeast genes, TIFSIB (ANBI) [lo, 161, COX% [ll, 121, CYC7 [131, HEM13 [141 and ERG11 [15] is negatively regulated by oxygen. These genes encode proteins functioning in various metabolic pathways including translation initiation (TIFSZB), mitochondrial respiratory chain (CYC7, COX%), heme (HEMZ3) and ergosterol (ERGll) biosynthesis. The oxygen repression in most of these genes appears to be mediated through the action of ROXl factor, the expression of which is heme dependent [ 17, 181. Deletion analysis and site-directed mutagenesis of TIF.51 B promoter pinpointed the upstream sequences which are most probably involved in the negative control of TIFSIB gene [19, 201. Similar sequences are also present in the upstream regions of the o...