Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria.

Abstract

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane‐spanning sided beta‐sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor‐like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high‐affinity sites and subsequent assembly to the mature form.

Role of the N‐ and C‐termini of porin in import into the outer membrane of Neurospora mitochondria

“…Briefly, restriction fragments of the Neurospora porin cDNA clone [18] were replaced by PCR products lacking either amino acid residues 2-12 or 3-20, to create A2-12porin and A3-20porin, respectively (Fig. 1).…”

“…Mitochondrial porins are predicted to traverse the outer membrane as a series of anti-parallel 13-strands (reviewed in [17]). The aminoterminal residues of porin have the potential to form an amphipathic c~-helix [17,18], and may lie at one surface of the membrane [19]. This region of the protein is not essential for either pore formation or voltage-dependent gating [20].…”

“…This region of the protein is not essential for either pore formation or voltage-dependent gating [20]. However, a potential role for this sequence as an import signal was suggested on the basis of its similarity to mitochondrial presequences and to the amino-terminus of Tom70 [18,21]. The N-termini of porins differ from cleavable presequences in that they possess both positively and negatively charged residues (Fig.…”

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

“…Cell-free synthesis of N. crassa precursor proteins in rabbit reticulocyte lysates (Pelham and Jackson, 1976) was carried out as described before using either poly(A)+ mRNA (Schleyer et al, 1982) or mRNA produced by in vitro transcription from cDNA cloned into transcription vectors (Pfanner et al, 1987a;Hart1 at al., 1987;Kleene et al, 1987). Desalting of postribosomal supernatants was performed by gel frltration using PD.10 columns (Pharmacra).…”

“…cDNA Cloning and DNA Sequencing Antibody screening of a N. crassa cDNA library (Kleene et al, 1987) in pEX vectors for 52 kd polypeptide clones was performed according to Stanley and Luzio (1984). One short cDNA insert, which was also positive in hybrid selection (Viebrock et al, 1982) of 52 kd mRNA, was used to screen a N. crassa cDNA library in pBR322 by colony hybridization.…”