Gertrud Mannhaupt

Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast

We identified a new, unique upstream activating sequence (5′‐GGTGGCAAA‐3′) in the promoters of 26 out of the 32 proteasomal yeast genes characterized to date, which we propose to call proteasome‐associated control element. By using the one‐hybrid method, we show that the factor binding to the proteasome‐associated control element is Rpn4p, a protein containing a C2H2‐type finger motif and two acidic domains. Electrophoretic mobility shift assays using proteasome‐associated control element sequences from two regulatory proteasomal genes confirmed specific binding of purified Rpn4p to these sequences. The role of Rpn4p to function as a transregulator in yeast is corroborated by its ability of stimulating proteasome‐associated control element‐driven lacZ expression and by experiments using the RPT4 and RPT6 gene promoters coupled to the bacterial cat gene as a reporter. Additionally, we found the proteasome‐associated control element to occur in a number of promoters to genes which are related to the ubiquitin‐proteasome pathway in yeast.

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m‐AAA protease, a protease complex consisting of multiple copies of the ATP‐dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP‐dependent metallopeptidase activity. While the m‐AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed ‘i‐AAA protease’. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane‐spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.

A nuclear mutation prevents processing of a mitochondrially encoded membrane protein in Saccharomyces cerevisiae.

Subunit II of cytochrome oxidase is encoded by the mitochondrial OXI1 gene in Saccharomyces cerevisiae. The temperature‐sensitive nuclear pet mutant ts2858 has an apparent higher mol. wt. subunit II when analyzed on lithium dodecylsulfate (LiDS) polyacrylamide gels. However, on LiDS‐6M urea gels the apparent mol. wt. of the wild‐type protein exceeds that of the mutant. Partial revertants of mutant ts2858 that produce both the wild‐type and mutant form of subunit II were isolated. The two forms of subunit II differ at the N‐terminal part of the molecule as shown by constructing and analyzing nuclear ts2858 and mitochondrial chain termination double mutants. The presence of the primary translation product in the mutant and of the processed form in the wild‐type lacking 15 amino‐terminal residues was demonstrated by radiolabel protein sequencing. Comparison of the known DNA sequence with the partial protein sequence obtained reveals that six of the 15 residues are hydrophilic and, unlike most signal sequences, this transient sequence does not contain extended hydrophobic parts. The nuclear mutation ts2858 preventing post‐translational processing of cytochrome oxidase subunit II lies either in the gene for a protease or an enzyme regulating a protease.

YTA10P, A MEMBER OF A NOVEL ATPASE FAMILY IN YEAST, IS ESSENTIAL FOR MITOCHONDRIAL FUNCTION

The yeast gene, YTA10, encodes a member of a novel family of putative ATPases. Yta10p, as deduced from the nucleotide sequence, is 761 amino acids in length (predicted molecular mass 84.5 kDa). The amino acid sequence of Yta10p exhibits high similarity to two other yeast proteins, Yta11 and Yta12, and to E coli FtsH. Several features of Yta10p are compatible with its localization in mitochondria. We report here that Yta10p is a yeast mitochondrial protein and that import is dependent on a membrane potential and accompanied by processing to a protein of approximately 73 kDa. Disruption of YTA10 leads to a nuclear petite phenotype and to a loss of respiratory competence, as shown by spectrophotometric measurement of the activities of respiratory complexes I–III and IV, respectively. These findings together with the high similarity of Yta10p to several ATP‐dependent proteases suggest that Yta10p is a mitochondrial component involved, directly or indirectly, in the correct assembly and/or maintenance of active respiratory complexes.

Characterization of the prephenate dehydrogenase-encoding gene, TYR1, from Saccharomyces cerevisiae

TYR1, the gene from Saccharomyces cerevisiae, which encodes prephenate dehydrogenase, one of the tyrosine biosynthetic enzymes, has been cloned by complementing a yeast tyr1 mutant strain. The DNA fragment containing the gene is part of a 45-kb cosmid clone which represents a region of chromosome II covering the genetically mapped tyr1 locus. The nucleotide sequence of a 3.1-kb region carrying the TYR1 gene and adjacent regions has been determined. The open reading frame contains 441 codons, corresponding to about 52.2 kDa for the encoded protein. The canonical NAD-binding domain is located within the first 45 amino acids of the protein. By primer extension, we show that there is one transcription start point. Presumably, the expression of TYR1 is not under the general GCN4 control. Instead, we find a dependence on the presence or absence of phenylalanine. These data were obtained by analysing CAT activity in constructs containing promoter fragments of TYR1 and the cat reporter gene.

A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast

Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay. To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used. We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants. To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region. We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low. Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast.

Genomic Evolution of the Proteasome System Among Hemiascomycetous Yeasts

Components of the proteasome-ubiquitin pathway are highly conserved throughout eukaryotic organisms. In S. cerevisiae, the expression of proteasomal genes is subject to concerted control by a transcriptional regulator, Rpn4p, interacting with a highly conserved cis-regulatory element, PACE, located in the upstream regions of these genes. Taking advantage of sequence data accumulated from 15 Hemiascomycetes, we performed an in silico study to address the problem of how this system might have evolved among these species. We found that in all these species the Rpn4p homologues are well conserved in terms of sequence and characteristic domain features. The “PACE patterns” turned out to be nearly identical among the Saccharomyces “sensu stricto” species, whereas in the evolutionary more distant species the putatively functional cis-regulatory motifs revealed deviations from the “canonical” PACE nonamere sequence in one or two nucleotides. Our findings suggest that during evolution of the Hemiascomycetes such slightly divergent ancestral motifs have converged into a unique PACE element for the majority of the proteasomal genes within the most recent species of this class. Likewise, the Rpn4 factors within the most recent species of this class show a higher degree of similarity in sequence than their ancestral counterparts. By contrast, we did not detect PACE-like motifs among the proteasomal genes in other eukaryotes, such as S. pombe, several filamentous fungi, A. thaliana, or humans, leaving the interesting question which type of concerted regulation of the proteasome system has developed in species other than the Hemiascomycetes.