Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.

Abstract

Molecular cloning and characterization of cytochrome c cDNA clones of Neurospora crassa wild‐type (74A) and a cytochrome c‐deficient mutant (cyc1‐1) are described. Southern blot analysis of genomic DNA indicates that only one cytochrome c gene exists in the N. crassa genome. The cDNA sequence of the wild‐type cytochrome c confirmed the previously determined protein sequence. Sequence analysis of the cyc1‐1 cDNA for cytochrome c revealed the presence of a larger open reading frame, owing to the presence of an unspliced intron in the 3′ end of the coding region. Splicing of this intron is obviously prevented due to the presence of two base exchanges in the highly conserved intron consensus sequences. Consequently, cyc1‐1 synthesizes apocytochrome c with an altered carboxy terminus, 19 amino acids longer than the wild‐type cytochrome c, with the final 27 amino acids being of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and, consequently, import into mitochondria. Thus, unlike other mitochondrial precursor proteins, where it has been demonstrated that the amino terminus alone is sufficient to target the protein to the mitochondria, an intact carboxy terminus is required for efficient import of apocytochrome c into mitochondria. This is independent confirmation for the view that the import pathway of cytochrome c is unique with respect to all other mitochondrial proteins studied to date.

Ssp1, a Site-specific Parvulin Homolog from Neurospora crassa Active in Protein Folding

“…Preparation of N. crassa Lysate-Cultures of N. crassa were grown as described (28). Hyphae from 10-liter cultures were frozen at Ϫ80°C for 1 h and then homogenized in a Waring blender for 10 min in the cold room with 2 volumes of lysate buffer (50 mM HEPES/NaOH, pH 7.8; 5 mM dithiothreitol; 1 mM EDTA; Boehringer complete protease inhibitors).…”

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

“…1.6 kb). For in vitro expression, cDNA insert 108 was cut with Ncol (deleting the entire 5'noncoding region including the dGdC tarls introduced by the cDNA cloning procedure) and cloned into transcription vector pGEM3 (Promega) (Stuart et al, 1987). For supercoil sequencing (Chen and Seeburg, 1985) full-length cDNA insert 108 was cloned into pUC19 in both orientations and shortened by the Exolll method (Henikoff, 1984).…”

Apocytochrome c: an exceptional mitochondrial precursor protein using an exceptional import pathway

“…The polypeptide sequence is changed from amino acid 102 onwards, resulting in an apocytochrome c which is 19 amino acids longer than the corresponding wild-type protein, thus the final 27 amino acids are of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and consequently for import into mitochondria [40]. Results, recently obtained would strongly suggest that alteration in the carboxy terminus of apocytochrome c, such as that observed in cycl-1 apocytochrome c, do not significantly affect the ability of the precursor to spontaneously insert into the mitochondrial membrane (Stuart and Neupert, unpublished results).…”

“…Cysteines number 14 and 17 (for N crassa) must be exposed to ihe intermembrane space for interaction with CCHL and subsequent heme linkage. The carboxy terminus of apocytochrome c has recently been shown to also be important for targeting of apocytochrome c to mitochondria [40]. As a consequence of a defect in cytochrome c mRNA splicing, a mutant of N crassa, cycl-1, synthesizes an apocytochrome c with an altered carboxy terminus.…”

“…Our current working hypothesis on the assembly pathway of cytochrome c is illustrated in figure 1 and can be summarized as follows. Cytochrome c is synthesized as a precursor protein known as apocytochrome c and does not contain an aminoterminal extension sequence, and thus is not proteolytically processed upon import into mitochondria [35][36][37][38][39][40]. No protease-sensitive components exist on the surface of mitochondria to mediate apocytochrome c binding and import [10].…”