Cells lacking Pcp1p/Ugo2p,a rhomboid-like protease required for Mgm1p processing,lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner,but remain competent for mitochondrial fusion

The dynamin-related GTPase, Mgm1p, is critical for the fusion of the mitochondrial outer membrane, maintenance of mitochondrial DNA (mtDNA), formation of normal inner membrane structures, and inheritance of mitochondria. Although there are two forms of Mgm1p, 100 and 90 kDa, their respective functions and the mechanism by which these two forms are produced are not clear. We previously isolated ugo2 mutants in a genetic screen to identify components involved in mitochondrial fusion [J. Cell Biol. 152 (2001) 1123]. In this paper, we show that ugo2 mutants are defective in PCP1, a gene encoding a rhomboid-related serine protease. Cells lacking Pcp1p are defective in the processing of Mgm1p and produce only the larger (100 kDa) form of Mgm1p. Similar to mgm1delta cells, pcp1delta cells contain partially fragmented mitochondria, instead of the long tubular branched mitochondria of wild-type cells. In addition, pcp1delta cells, like mgm1delta cells, lack mtDNA and therefore are unable to grow on nonfermentable medium. Mutations in the catalytic domain lead to complete loss of Pcp1p function. Similar to mgm1delta cells, the fragmentation of mitochondria and loss of mtDNA of pcp1delta cells were rescued when mitochondrial division was blocked by inactivating Dnm1p, a dynamin-related GTPase. Surprisingly, in contrast to mgm1delta cells, which are completely defective in mitochondrial fusion, pcp1delta cells can fuse their mitochondria after yeast cell mating. Our study demonstrates that Pcp1p is required for the processing of Mgm1p and controls normal mitochondrial shape and mtDNA maintenance by producing the 90 kDa form of Mgm1p. However, the processing of Mgm1p is not strictly required for mitochondrial fusion, indicating that the 100 kDa form is sufficient to promote fusion.     

Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp).

The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.     

Molecular cloning and characterization of Mycobacterium bovis BCG pcp gene encoding pyrrolidone carboxyl peptidase.

The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced. The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da. The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. A multiple sequence alignment revealed highly conserved domains. The BCG pcp gene was overexpressed in Escherichia coli. The Pcp was purified to homogeneity. The recombinant protein was further confirmed by an enzymatic assay.     

A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression.

Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.     

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.     

Negative Dominant Mutations of the uidR Gene in ESCHERICHIA COLI: Genetic Proof for a Cooperative Regulation of uidA Expression

The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.