Antonella Ragnini

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 theMRS2 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.

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.

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°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.

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 were 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.

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. Unexpeciedly, the MRS6 gene product also affects mitochondrial functions as revealed by the facts that highcopy 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.

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.

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. Copyright

REP-Mediated Protein Prenylation

Addition of isoprenoids to polypeptides is a common post-translational modification occurring in yeast, animal and plant cells. Isoprenoid precursors are derived from the mevalonate pathway and are incorporated into many cellular products such as sterols, heme A, ubiquinone, dolichols, carotenoids and prenylated proteins (Goldstein and Brown, 1990).