PubMed Health⌕ Search

Biomedical subjects

A Ragnini

Publications and source records attributed to A Ragnini.

18 recordsLinked to original sources
PubMed2001

Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells.

Farnesyl diphosphate (FPP), an intermediate of the sterol biosynthetic pathway, is used by farnesyl transferase to farnesylate, among others, the Ras proteins, and by geranylgeranyl diphosphate synthase to produce geranylgeranyl diphosphate (GGPP). GGPP is then transferred by geranylgeranyl transferase II (GGTase II) to Rab/Ypt members of the Ras superfamily known to be required at all stages of vesicle transport in both mammals and yeast. Formation of a complex between a Rab/Ypt protein and an accessory protein named the Rab escort protein (REP) is a prerequisite for GGTase II substrate recognition. Little is known about the factors that regulate GGTase II activity in living cells but, based on available data, it seems possible that vesicle transport in higher eukaryotes is regulated by the levels of prenylated Rab/Ypt proteins in the cells. Here we show that the levels of REP play an important role in regulating GGTase II activity in yeast cells if sufficient substrates are present. Moreover, overexpression of REP causes, directly or indirectly, an increased level of Ypt substrates available for prenylation, which in turn leads to the depletion of the GGPP pool in the cell. Overall our data suggest that the levels of REP and the availability of GGPP play a role in regulating Ypt protein prenylation.

Alkyl and Aryl Transferases↗
PubMed2000

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance.

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane.

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.

Bacterial Proteins↗
PubMed1997

The yeast Rab escort protein binds intracellular membranes in vivo and in vitro.

In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria.

The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed.

Amino Acid Sequence↗
PubMed1994

The yeast protein Mrs6p, a homologue of the rabGDI and human choroideraemia proteins, affects cytoplasmic and mitochondrial functions.

MRS6 is a newly-identified gene in the yeast Saccharomyces cerevisiae. Its product Mrs6p shows significant homology to the mammalian GDP dissociation inhibitor (GDI) of Rab/Ypt-type small G proteins and to the human choroideraemia protein (CHM), the component A of Rab-specific GGTase II. The interaction of Mrs6p with G proteins is indicated by our observation that the MRS6 gene suppresses the effect of a temperature-sensitive ypt1 mutation. Disruption of the MRS6 gene is lethal to haploid yeast cells. This is consistent with the notion that Mrs6p is interacting with Rab/Ypt-type small G proteins, which are known to have essential functions in vesicular transport. Unexpectedly, the MRS6 gene product also affects mitochondrial functions as revealed by the facts that high-copy numbers of MRS6 (1) suppress the pet- phenotype of mrs2-1 mutant strains and (2) cause a weak pet- phenotype in wild-type strains. We conclude from these results that the MRS6 gene product has a vital function in connection with Rab/Ypt-type proteins in the cytoplasm and, in addition, affects mitochondrial functions.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Mrs6p, the yeast homologue of the mammalian choroideraemia protein: immunological evidence for its function as the Ypt1p Rab escort protein.

The Saccharomyces cerevisiae MRS6 gene belongs to the same gene family as that responsible for the mammalian Rab escort protein (REP) and the Rab GDP dissociation inhibitor protein (GDI). Both REP and GDI are regulators of the Ras-related small G-proteins Rab/YPT1 which are involved in intracellular vesicular trafficking in yeast and in mammals. Here we characterize an antiserum directed against Mrs6p and show that it specifically inhibits the geranylation of the YPT1 protein in an in vitro assay. These findings provide direct evidence for the role of Mrs6p as the REP component of the yeast Rab geranylgeranyl transferase enzyme.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Ordered processing of the polygenic transcripts from a mitochondrial tRNA gene cluster in K. lactis.

In Saccharomyces cerevisiae, transcription of the mitochondrial genome starts at multiple initiation sites and is followed by the processing of multigenic transcripts at the 5' and 3' termini of tRNA sequences and in some intergenic regions. We have used a comparative approach to investigate the structure and function of the latter processing sites. We present here an analysis of the transcripts of a cluster of tRNA genes from the mitochondrial genome of Kluyveromyces lactis. The gene order of this cluster is the same as that of the cluster in S. cerevisiae but the sequence of the intergenic regions is different. A detailed analysis of transcripts has been performed using S1 mapping and primer extension techniques. The results can be summarized as follows: (1) transcription of the cluster very probably starts at initiation sites having the nonanucleotide sequence TTATAAGTA (which acts as a promoter in S. cerevisiae) and yields polygenic transcripts; (2) processing of these transcripts seems to occur through an ordered pathway of endonucleolytic events in which some tRNA sequences are preferentially excised and some endonucleolytic cuts occur more readily than others; (3) in two intergenic regions, strong signals indicate the existence of processing events. The sequences around these sites are similar in sequence and localization to S. cerevisiae intergenic processing sites, indicating a possible functional importance in maintaining a conserved order of tRNA genes in different species of yeasts.

Amino Acid Sequence↗
PubMed1993

A multitude of suppressors of group II intron-splicing defects in yeast.

Disruption of the nuclear MRS2 gene (mrs2-1 mutation) causes a strong pet- phenotype in strains with mitochondrial group II introns, and a leaky pet- phenotype in strains without group II introns. MRS3 and MRS4, the genes for two mitochondrial-solute carrier proteins, can suppress both phenotypes when present in high-copy-number plasmids. In order to search for further multicopy suppressors of the mrs2-1 mutant phenotype, an yeast genomic DNA library, MW90, was constructed in YEp351 from a strain deleted for the MRS2, MRS3 and MRS4 genes. Ten different Sau3A DNA fragments that act as multicopy suppressors of the mrs2-1 respiratory-deficient phenotype were isolated from this library. Some of the newly isolated genes suppress the pet- phenotypes of mrs2-1 cells in strains with and without mitochondrial group II introns. Other genes, however, are suppressors only for the mitochondrial intron-less strains. This supports the notion that the MRS2 gene product is bifunctional i.e., it is essential for the splicing of group II introns and is also involved in processes of mitochondrial biogenesis other than RNA splicing.

Genes, Fungal↗
PubMed1991

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Adenosine Triphosphatases↗
PubMed1991

Mitochondrial genome of Saccharomyces douglasii: genes coding for components of the protein synthetic apparatus.

Mitochondrial genes coding for some components of the protein synthetic apparatus in S. douglasii have been studies in detail. A region containing stretches of high homology to the S. cerevisiae tRNA synthesis locus (TSL) and the tRNA(fmet) gene has been identified and sequenced. The organization of this region was very similar to that present in S. cerevisiae, including the presence of a possible transcription starting signal. The S. douglasii TSL gene is shorter due to several deletions which, however, do not involve the regions coding for RNA domains know to be required for the catalytic activity of mitochondrial RNAse P. The S. douglasii LSU rRNA gene has been shown to contain a typical group I intron highly homologous to its S. cerevisiae counterpart, except for the absence of the open reading frame which in S. cerevisiae codes for I-SceI endonuclease.

Base Sequence↗
PubMed1989

Genetic instability of an oligomycin resistance mutation in yeast is associated with an amplification of a mitochondrial DNA segment.

In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable. In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant. When the mutant was grown in the absence of the drug, the resistance was rapidly lost. This character showed a typical cytoplasmic inheritance. The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA. The amplified molecules were co-replicating with the wild type genome in the mutant cells. The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA. The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl). Modified processing of these tRNAs was detected in the mutant. A possible mechanism by which these events could lead to drug resistance is discussed.

Base Sequence↗
PubMed1989

A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing.

A temperature sensitive mutation mapping in the tRNA region of the mitochondrial genome of S. cerevisiae has been found to abolish 3' processing of tRNA(asp). Mutant cells grown for a few generations at the non-permissive temperature were found to specifically lack mature tRNA(asp) and to accumulate 3' unprocessed precursors of this tRNA. The accumulation of precursors of other mitochondrial tRNAs was also observed under the same conditions. After longer incubation times, a generalized decrease of mitochondrial transcripts could be observed. The mutation was genetically mapped in a limited region surrounding the tRNA(asp) gene and found, by sequencing, to consist of a C- greater than T transition at position 61 of the tRNA(asp) gene.

Base Sequence↗
PubMed1989

Analysis of the regions coding for transfer RNAs in Kluyveromyces lactis mitochondrial DNA.

The major regions coding for the transfer RNA genes in the mitochondrial DNA of K. lactis were studied. Twenty one, out of a supposed twenty four tRNA genes were identified and localized with respect to other mitochondrial genes. Most of the tRNA genes were found in a cluster downstream of the large ribosomal RNA gene. The order of a few groups of genes is conserved with respect to S. cerevisiae and T. glabrata. The highly diverged intergenic sequences contained a large number of guanine-cytosine clusters which frequently formed long palindromic sequences.

Base Composition↗
PubMed1988

Mitochondrial DNA of the yeast Kluyveromyces: guanine-cytosine rich sequence clusters.

Mitochondrial DNA from the yeast Kluyveromyces marxianus var. lactis (K.lactis) is a circular molecule of 39 kilobase-pairs. A genetic and physical map was constructed. We found that this genome contained a large number of guanine-cytosine (GC)-rich sequence clusters, many of which are characterized by the presence of SacII restriction sites (CCGCGG). The primary sequence of the GC clusters often showed a palindromic structure. These GC clusters were present in several varieties of K.marxianus, but not in others. The presence of these clusters is a major feature that distinguishes K.lactis strains from those of K.marxianus var. marxianus (including K.fragilis).

Base Sequence↗
PubMed1987

Analysis of transcripts of the major cluster of tRNA genes in the mitochondrial genome of S. cerevisiae.

The transcripts of a 6Kbp region of the mitochondrial genome of S. cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts. The study was performed in the w.t. strain D273-10B and in several rho- mutants retaining different, partially overlapping portions of the studied region; the mutants accumulate incompletely-processed precursors of tRNAs due to the absence of the tRNA synthesis locus. Results show the presence in the region of four sites at which initiation occurs at a consensus nonanucleotide ATTATAAGTA (or a minor variant of the same); however different initiation sites are used in different strains, and several differences as compared to initiation in vitro can also be observed. Termini arising by processing are often localized at AATATAA or AATATATTTT sequences localized immediately adjacent to a G+C cluster or a tRNA sequence.

Base Sequence↗