The primary structure of cytochrome c1 from Neurospora crassa

Abstract

The primary structure of the cytochrome c1 subunit of ubiquinol‐cytochrome‐c reductase from mitochondria of Neurospora crassa was determined by sequencing the cDNA of a bank cloned in Escherichia coli. From the coding region the sequence of 332 amino acids, corresponding to the molecular mass of 36496 Da, was derived for the precursor protein. The mature protein, the N terminus of which was previously sequenced [Tsugita et al. (1979) in Cytochrome oxidase (King, T. E. et al., eds) pp. 67–77, Elsevier, New York], consists of 262 amino acids and has the molecular mass of 29 908 Da including the heme. The sequence contains an N‐terminal hydrophilic part of 211 residues, which carries the heme, a hydrophobic stretch of 15 residues, which is assumed to anchor the protein to the membrane, and a C‐terminal hydrophilic part of 36 residues. The N‐terminal presequence of 70 amino acids contains 9 positive charges but only 1 negative charge and is characterized by a stretch of 20 uncharged residues.

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

“…In this report we describe the identification, sequencing and expression of porin cDNA from N. crassa. The deduced amino acid sequence reveals that the primary structure of porin is not strongly conserved between N. crassa and Saccharomyces cerevisiae, in contrast to that of other mitochondrial proteins, such as ADP/ATP carrier (Arends and Sebald, 1984;Adrian et al, 1986), cytochrome cl (Romisch et al, 1987;Sadler et al, 1984), cytochrome c (Heller and Smith, 1966;Lederer and Simon, 1974;Stuart et al, 1987;Boss et al, 1981) and fl-subunit of F1-ATPase (Rassow, Neupert and Tropschug, in preparation; Takeda et al, 1985). Despite this fact, the secondary structure of the porins from N. crassa and S. cerevisiae seem to be significantly conserved as sided ,B-sheets which traverse the membrane.…”

Early steps in mitochondrial protein import: Receptor functions can be substituted by the membrane insertion activity of apocytochrome c

“…For the construction of the plasmids encoding the fusion protein SC,-c (Figure l), the EcoRI-Hindlll fragment of N. crassa cytochrome cr DNA containing the 5' noncoding region and the amino-terminal half of the cytochrome cr protein (Romisch et al, 1987) was cloned into the plasmid pMa5-8 (Kramer et al, 1984). Oligonucleotide sitedirected mutagenesis (essentially as described by Mijller and Zimmermann, 1988) was performed to introduce a unique Pvull site in the cytochrome cl cDNA sequence by changing a C residue for an A residue at a position corresponding to codon 34.…”

“…It is interesting in this regard to compare the import of the two c-type cytochromes in mitochondria, namely cytochromes c and cl, both of which are located on the outer surface of the inner membrane. The precursor of cytochrome c, (PC,) contains a long amino-terminal prepiece that is processed in two steps upon import into mitochondria (Teintze et al, 1982;Gasser et al, 1982;Ohashi et al, 1982;Schleyer and Neupert, 1985;Sadler et al, 1984;Romisch et al, 1987). After initial binding to protease-sensitive surface receptors, PC, inserts into the outer membrane (Pfaller et al, 1988, and this work) and is then imported via contact sites (Schleyer and Neupert, 1985) into the mitochondrial matrix where it is processed to its intermediate form (iC,).…”

Two Distinct and Independent Mitochondrial Targeting Signals Function in the Sorting of an Inner Membrane Protein, Cytochromec 1

“…Cytochrome c 1 , a subunit of the cytochrome bc 1 complex of the respiratory chain, is anchored to the inner membrane in an N out -C in orientation via a single transmembrane segment near its COOH terminus (7). It is synthesized as a precursor protein, precytochrome c 1 which contains an NH 2 -terminal cleavable bipartite presequence (8,9). The first part of this presequence is a mitochondrial targeting signal which becomes proteolytically removed by MPP in the matrix to generate an intermediate size cytochrome c 1 (intermediate size Cytc 1 ).…”