Rpm2p: separate domains promote tRNA and Rpm1r maturation in Saccharomyces cerevisiae mitochondria.

Rpm2p is a protein subunit of yeast mitochondrial RNase P and is also required for the maturation of Rpm1r, the mitochondrially-encoded RNA subunit of the enzyme. Previous work demonstrated that an insertional disruption of RPM2, which produces the C-terminally truncated protein Rpm2-DeltaCp, supports growth on glucose but cells lose some or all of their mitochondrial genome and become petite. These petites, even if they retain the RPM1 locus, lose their ability to process the 5'-ends of mitochondrial tRNA. We report here that if strains containing the truncated RPM2 allele are created and maintained on respiratory carbon sources they have wild-type mitochondrial genomes, and a significant portion of tRNA transcripts are processed. In contrast, precursor Rpm1r transcripts accumulate and mature Rpm1r is not made. These data show that one function of the deleted C-terminal region is in the maturation of Rpm1r, and that this region and mature Rpm1r are not absolutely required for RNase P activity. Finally, we demonstrate that full activity can be restored if the N-terminal and C-terminal domains of Rpm2p are supplied in trans.

Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase.

RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa(3) cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.

Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

Evidence for a phosphoenzyme intermediate formed during catalysis by pyridoxal phosphatase from human erythrocytes.

Pyridoxal phosphatase purified from human erythrocytes catalyzes the dephosphorylation of pyridoxal phosphate (PLP) and pyridoxine phosphate. The enzyme had phosphotransferase activity and transferred 20-25% of the phosphoryl group from either substrate to ethanol. Incubation of the enzyme with [32P]PLP, followed by quenching in acid, resulted in trapping 0.14-0.24 mol of 32P per mol of subunit. The incorporation of 32P was not due to Schiff base formation. Phosphorylation of the enzyme by [32P]PLP required catalysis by the enzyme and did not occur in the presence of excess pyridoxine phosphate or with denatured enzyme. The phosphoenzyme intermediate was relatively acid stable and very labile at high pH or in the presence of hydroxylamine. Woodward's reagent K, which specifically modifies acidic amino acid residues, inactivated the phosphatase in a concentration- and time-dependent manner which followed pseudo-first-order kinetics. Substrates or Pi protected the enzyme from inactivation. It is concluded that PLP phosphatase catalyzes the hydrolysis of PLP by forming a covalent phosphoenzyme intermediate and the intermediate may be an acylphosphate. The 32P-labeled phosphatase was digested with pepsin, and two radioactive peaks were isolated by reversed-phase chromatography. However, definitive sequences were not obtained.

Kinetic analysis and chemical modification of vitamin B6 phosphatase from human erythrocytes.

The specificity and active site properties of vitamin B6 phosphatase purified from human erythrocytes were studied by kinetic analyses with vitamin B6 compounds and derivatives and chemical modification with group-specific reagents. The kinetic constants for pyridoxal phosphate (PLP), 4-pyridoxic acid phosphate, pyridoxine phosphate, and pyridoxamine phosphate were determined from pH 5 to 9. The values of Vmax/Km and pKm were highest for PLP and 4-pyridoxic acid phosphate and lowest for pyridoxamine phosphate. Vmax/Km and pKm for the four substrates were maximum between pH 6 and 8. Ionizable groups with pKa values about 6 and 8 affected substrate binding to the enzyme. Vmax values for all the substrates gradually decreased with increasing pH. The enzyme also catalyzed the dephosphorylation of 4'-secondary amine derivatives of vitamin B6-phosphate. The phosphatase had greatest catalytic efficiency with substrates that contained a negatively charged group on the 4'-position of the pyridine ring. It is concluded that there are one or two positively charged groups at the active site of the enzyme that interact with the substrate's phosphate ester and 4'-substituent. The phosphatase was inactivated by phenylglyoxal, and PLP protected the enzyme against this inactivation. Phenylglyoxal did not modify Lys or Cys residues or an alpha-amino group since the enzyme's NH2 terminus is blocked, and it did not affect the quaternary structure of the phosphatase. The enzyme was inactivated by the incorporation of 1 mol of phenylglyoxal/subunit. Diethylpyrocarbonate inactivated the enzyme by reacting with a group with a pKa of 6.7, and pyridoxine phosphate protected the enzyme against this inactivation. These data suggest that Arg and His residues are at or near the active site and may play roles in substrate binding and/or catalysis.