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.     

Genetic evidence for selective degradation of RNA polymerase subunits by the 20S proteasome in Saccharomyces cerevisiae.

scs32 was isolated as an extragenic suppressor of a temperature-sensitive (ts) mutation (rpo26-31) in the gene encoding Rpo26p, a subunit common to yeast nuclear RNA polymerases (RNAPs). rpo26-31 also confers inositol auxotrophy, inhibits the assembly of RNAPI and RNAPII and reduces the steady-state level of Rpo26p and the largest subunit of RNAPI (Rpo11p or A190p) and RNAPII (Rpo21p). rpo26-31p accumulated to wild-type levels in the scs32 strain; nevertheless, the amount of assembled RNAPII remained at a reduced level at high temperature. Hence, scs32 only partially suppressed the ts phenotype and was unable to suppress the Ino-phenotype of rpo26-31. SCS32 is identical to PUP3, which encodes a subunit of the yeast proteasome. scs32 was able to suppress the phenotype of other ts alleles of RPO26, all of which reduce the steady-state level of this subunit. However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p. These observations suggest that the stability of non-functional or unassembled forms of Rpo26p and Rpo21p are regulated independently.     

Unique features of plant mitochondrial sulfhydryl oxidase

The yeast and human mitochondrial sulfhydryl oxidases of the Erv1/Alr family have been shown to be essential for the biogenesis of mitochondria and the cytosolic iron sulfur cluster assembly. In this study we identified a likely candidate for the first mitochondrial flavin-linked sulfhydryl oxidase of the Erv1-type from a photosynthetic organism. The central core of the plant enzyme (AtErv1) exhibits all of the characteristic features of the Erv1/Alr protein family, including a redox-active YPCXXC motif, noncovalently bound FAD, and sulfhydryl oxidase activity. Transient expression of fusion proteins of AtErv1 and the green fluorescence protein in plant protoplasts showed that the plant enzyme preferentially localizes to the mitochondria. Yet AtErv1 has several unique features, such as the presence of a CXXXXC motif in its carboxyl-terminal domain and the absence of an amino-terminally localized cysteine pair common to yeast and human Erv1/Alr proteins. In addition, the dimerization of AtErv1 is not mediated by its amino terminus but by its unique CXXXXC motif. In vitro assays with purified protein and artificial substrates demonstrate a preference of AtErv1 for dithiols with a defined space between the thiol groups, suggesting a thioredoxin-like substrate.     

Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion

In yeast, mitochondrial fusion requires Ugo1p and two GTPases, Fzo1p and Mgm1p. Ugo1p is anchored in the mitochondrial outer membrane with its N terminus facing the cytosol and C terminus in the intermembrane space. Fzo1p is also an outer membrane protein, whereas Mgm1p is located in the intermembrane space. Recent studies suggest that these three proteins form protein complexes that mediate mitochondrial fusion. Here, we show that the cytoplasmic domain of Ugo1p directly interacts with Fzo1p, whereas its intermembrane space domain binds to Mgm1p. We identified the Ugo1p-binding site in Fzo1p and demonstrated that Ugo1p-Fzo1p interaction is essential for the formation of mitochondrial shape, maintenance of mitochondrial DNA, and fusion of mitochondria. Although the GTPase domains of Fzo1p and Mgm1p regulate mitochondrial fusion, they were not required for association with Ugo1p. Furthermore, we found that Ugo1p bridges the interaction between Fzo1p and Mgm1p in mitochondria. Our data indicate that distinct regions of Ugo1p bind directly to Fzo1p and Mgm1p and thereby link these two GTPases during mitochondrial fusion.     

Structure-function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.