1. The potassium channel beta‐subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1‐110. 2. Kv beta 1.1 was co‐expressed in Xenopus oocytes with various other potassium channel alpha‐subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2‐28 and Kv4.1 channels could be observed. 3. The second member of the beta‐subunit subfamily, Kv beta 2, had a shorter N‐terminal end, accelerated inactivation of the A‐type channel Kv 1.4, but did not induce inactivation when co‐expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co‐assembles with Kv alpha‐subunits, the N‐terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N‐terminus of Kv beta 2. The chimaeric beta‐subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha‐subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2‐28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about ‐10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans.

The proteome analysis by 2-DE is one of the most potent methods of analyzing the complete proteome of cells, cell lines, organs and tissues in proteomics studies. It allows a fast overview of changes in cell processes by analysis of the entire protein extracts in any biological and medical research projects. New instrumentation and advanced technologies provide proteomics studies in a wide variety of biological and biomedical questions. Proteomics work is being applied to study antibiotics-resistant strains and human tissues of various brain, lung, and heart diseases. It cumulated in the identification of antigens for the design of new vaccines. These advances in proteomics have been possible through the development of advanced high-resolution 2-DE systems allowing resolution of up to 10 000 protein spots of entire cell lysates in combination with protein identification by new highly sensitive mass spectrometric techniques. The present technological achievements are suited for a high throughput screening of different cell situations. Proteomics may be used to investigate the health effects of radiation and electromagnetic field to clarify possible dangerous alterations in human beings.

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51 -561. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new. [ 5 , 6 ] . About a half of the yeast mitoribosoma1 proteins characterized so far show a high degree of similarity to eubacterial ribosomal proteins, supporting the notion that mitochondria indeed originate from eubacteria-like organisms. However, the other half of the mitoribosomal proteins show no apparent similarity to any known ribosomal proteins of eubacterial or eukaryotic (cytoplasmic) origin. Considering the fact that mitoribosomes appear to contain more proteins than Escherichiu coli ribosomes [I], those proteins which are not similar to any known ribosomal proteins might have been recruited during the course of evolution from other sources unrelated to ribosomes. There are nonetheless many E. coli ribosomal proteins whose homologues have not yet been identified in the yeast mitoribosome: these proteins include L7L12 and L2.5 which have been shown to be present in all eubacterial ribosomes studied so far and are implicated as being functionally important. Therefore, from an evolutionary point of view, it is of great interest to investigate how many proteins are present in the mitoribosome of S. cerevisiae and with which eubacterial ribosomal proteins they have evolutionary relations. Now that the nucleotide sequence of the entire genome of S.cerevisiae has been determined, it is possible to identify the genes encoding mitoribosomal proteins by computer search if the amino acid sequences of their peptides, even as short as several residues, are known. During the course of our amino acid sequence analysis of yeast mitoribosomal proteins, we obtained fragmentary sequence data for many large subunit and several small subunit proteins. Therefore, we used the data to search for likely yeast genes encoding mitoribosomal proteins as will be described below. MATERIALS AND METHODSMitochondria were prepared from cells of S. cerevisiae strain 07173 (da; wild type), and mitoribosomal proteins were extracted from the small and large subunits separated by sucrose gradient centrifugation as described [3].About . 5 A,,, units of TP50 (total large subunit proteins), dialyzed against 5 % acetic acid, we...

No abstracts.

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.Intact mitochondrial protein biosynthesis has been shown to be indispensable for the maintenance of mitochondrial DNA in yeast (1). Nearly all of the mitochondrial ribosomal proteins (MRPs) 1 investigated so far are essential for proper mt protein synthesis (2). Knock-out mutants of yeast MRP genes lose their respiratory capacity and change to Ϫ or o mt genetic status by successive losses of mt DNA (1). In higher eukaryotes, the knowledge about comparable functions of MRPs is only rudimentary, since only a few MRPs have been characterized on the molecular level. The protein composition of mammalian mt ribosomes has been studied extensively (3-5). Some properties of mt ribosomes such as structure (6), binding of nucleotides and RNA (7-11), and interaction with different factors have been studied (12)(13)(14). However, only 3 of the approximately 80 -100 different human MRPs have been described at the molecular level so far. MRL3, which is the EcoL3 counterpart in human mt ribosomes, was identified as an overexpressed r-protein in Mahlavu hepatomic cells (15). Later, it was postulated to be a true MRP by virtue of its sequence similarity to the corresponding yeast MRP YmL9 (16). MRPL12 was identified as a delayed-early response gene similar in sequence to the Escherichia coli L7/L12 r-protein (17). The metazoan mitochondrial counterpart of EcoS12 has been characterized in Drosophila, human, and mouse (18,19). In Drosophila a mutation of mt S12 causes abnormal behavior. This is the first case reported so far of affection of the status of an animal by an MRP mutation (18). Diseases affecting mitochondria are known in humans, and are caused by nuclear mutations responsible for the loss of mt DNA as a secondary effect by a so far unknown mechanism (20, 21). Mutations of MRPs are good candidates affecting mt genetic and/or physiological status. To characterize mammalian MRPs and to compare their biochemical properties with that of their (essential) counterparts, e.g. of yeast, we identified several mammalian MRPs and their corresponding gene sequences. We used N-terminal sequence inform...

Proteins of the small and large subunits of mitcchondrial ribosomes from the yeast Succharomyces cerevisiae were isolated and characterized by two-dimensional gel electrophoresis. Ribosomal proteins of the large subunit were separated by reverse-phase HPLC and up to 37 amino acid residues of the N-terminal sequences of L3, L4, L9 and L31 were determined. No significant homology to ribosomal protein sequences so far determined from other organisms was found.

We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochond~a by direct amino acid micro-sequencing.The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides eesponsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found. a programme for the isoiation and sequencing of mitochondrial r-proteins 121. N-Terminal sequence data were used for cloning of the nuclear genes by oligonucleotide hybridisation [3,4]. In this way several nuclear genes coding for mitochondrial r-proteins have been cloned and anaIysed [3-71, and it was found that 3 of the sequences are significantly related to eubacterial r-proteins. More generally, yeast nuclear genes coding for mitochondrial proteins were cloned by genetic complementation of pet mutants (for review see [8]). Pet mutant genes often affect gene products that are either directly involved in the oxidative metabolism of mitochondria or are necessary for expression of its activity [9]. For example, the PET-genes MRPl and MRPZ were found to code for mitochondria1 r-proteins [IO]. It is to be expected that several more MRPs will be analysed, since most of the mitochondrial r-proteins are essential for the translational activity of mito-ribosomes [4-7,10,1 I]. Recently, McMullin et al. [ll] identified PET123 as a component of the small ribosomai subunit in yeast. Nevertheless, in most cases it is difficult to define the exact function of the PET-genes, and additional biochemical analysis is necessary to assign them a function. Even with more data this function might remain unknown [12]. Therefore, N-terminal sequences of mitochondrial r-proteins could be a fruitful basis not only for cloning of the corresponding genes, but also for the identification and/or assignment of other PET-gene products in the future.In the present study we show the results of extended N-terminal amine acid sequence analysis of 26 mitochondrial r-proteins, Proteins MRP7, YMR-26 and 51