Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii

Abstract

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.

FIG. 1

Schematic representation of the B. emersonii α-MPP gene. The diagram shows a partial restriction map of the sequenced region (2.2 kb) of the genomic clone SacI-SacI. The open rectangles represent the coding region of the α-MPP gene, the stippled area depicts an intron (112 bp), and the lines represent upstream and downstream regions of the gene. The position of the PCR-amplified fragment, used as a probe to clone the α-MPP gene, is indicated.

FIG. 2

Nucleotide sequence of the B. emersonii α-MPP gene and the deduced amino acid sequence. Capital letters indicate deoxynucleotides in exons or sequences upstream and downstream of the coding region of the gene; lowercase letters show the deoxynucleotides in the intron. Nucleotide +1 denotes the A of the ATG of the initiator methionine. Residues preceding it are indicated by negative numbers. The deduced amino acid sequence is shown below the nucleotide sequence. The arrows (↓) indicate putative signal sequence cleavage sites. A probable polyadenylation signal is shown in bold.

FIG. 3

Comparison of the α-MPP from different organisms. The amino acid sequence alignment of α-MPP from B. emersonii (Be [this report]), S. cerevisiae (Sc [21]), rat (23), potato (7), and N. crassa (Nc [42]) is shown. Identical amino acids are indicated by asterisks. Conserved residues are indicated by black dots. The four cysteine residues conserved in B. emersonii, S. cerevisiae, and N. crassa are indicated by arrows. The putative metal-binding site motif is delimited by a gray rectangle. Gaps in the alignment are marked by dashes within the sequences. Sequence comparison was performed with the program ClustalV (1).

FIG. 4

Transcription start site of the α-MPP gene. (A) Primer extension mapping of the transcription start site. An 18-nt primer, complementary to nt +146 to +163, was 5′ end labeled with [γ-³²P]ATP and hybridized to 50 μg of total RNA from B. emersonii vegetative cells (lane 1), zoospores (lane 2), and germling cells (lane 3). The hybrids were then extended with reverse transcriptase, and the extension products were resolved by denaturing gel electrophoresis and autoradiography. The sequencing ladder was generated with the same 18-nt oligonucleotide as a primer and M13mp19 containing the 5′ end of the α-MPP gene (coding strand). (B) Nucleotide sequence of the 5′ region of the α-MPP gene. Nucleotide +1 denotes the A of the ATG encoding the initiator methionine. The underlined region is complementary to the oligonucleotide used for the primer extension experiment. The predicted transcription start point is indicated by an arrow. The putative core sequences representing the binding sites for the TATA-binding protein (TATA box), Sp1 (GC box), CTF/NF1 (CCAAT box), and helix-loop-helix transcription factor (E box) are indicated.

FIG. 5

α-MPP mRNA levels during B. emersonii development. Total RNA (10 μg/lane) isolated from cells at different stages of the B. emersonii life cycle were subjected to electrophoresis in a formaldehyde-agarose gel, transferred to Hybond N+ membrane, and probed with ³²P-labeled PCR-amplified fragment. Lanes: 1 to 5, sporulating cells 0, 60, 90, 120, and 180 min after starvation, respectively; 6, zoospores; 7 and 8, germinating cells 45 and 90 min after inoculation in DM3 medium. As a control, the same blot was hybridized to a ³²P-labeled rat GAPDH cDNA. (A) Autoradiograms of the blot. (B) Relative amount of α-MPP mRNA, determined by scanning of the autoradiograms, normalized with respect to the GAPDH signal.

FIG. 6

Subcellular localization of α-MPP from B. emersonii. Total zoospore extract (lanes 1) was centrifuged at 100,000 × g, resulting in a soluble fraction (lanes 2) and a particulate fraction (lanes 3). The particulate fraction was then subjected to alkaline treatment with 0.1 M Na₂CO₃, and the suspension was centrifuged at 100,000 × g for 10 min, giving rise to an insoluble fraction (lanes 4) and a soluble fraction (lanes 5). Equal amounts of protein of each fraction (10 μg/lane) were analyzed by SDS-PAGE and Western blotting with anti-B. emersonii α-MPP antiserum, anti-B. emersonii β-MPP antiserum, anti-B. emersonii P-type ATPase antiserum, anti-HSP60 antiserum (Sigma), and antiserum against subunit VII (Sub VII) of S. cerevisiae cytochrome c reductase complex. The blots were developed with the ECL kit (Amersham). The molecular masses (kDa) of the recognized polypeptides are indicated.