The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA.

Abstract

The proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa is an extremely hydrophobic protein of 81 amino acid residues, which is imported into mitochondria as a precursor of mol. wt. 15 000. The primary structure of the imported form has now been determined by isolating and analyzing cDNA clones of the preproteolipid mRNA. An initial cDNA clone was identified by hybridizing total polyadenylated RNA to pooled cDNA recombinant plasmids from an ordered clone bank and subsequent cell‐free translation of hybridization‐selected mRNA. Further preproteolipid clones were identified at a frequency of 0.2% by colony filter hybridization. One isolated cDNA represented the major part of the preproteolipid mRNA. The nucleotide sequence showed 243 bases corresponding to the mature proteolipid and, in addition, 178 bases coding for an amino‐terminal presequence . Non‐coding sequences of 48 bases at the 5′ end and of 358 bases at the 3′ end plus a poly(A) tail were determined. The long presequence of 66 amino acids is very polar, in contrast to the lipophilic mature proteolipid, and includes 12 basic and no acidic side chains. It is suggested that the presequence is specifically designed to solubilize the proteolipid for post‐translational import into the mitochondria.

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

“…The precipitate was subjected to electrophoresis; mature subunit 9 was eluted from the gel (40,000 cpm) and subjected to 25 steps of solid-phase Edman degradation (Wachter et al, 1973). The amino acid sequence of Neurospora subunit 9 is shown (Viebrock et al, 1982); +1 marks the amino terminus of mature subunit 9. Radioactivity is released with methionines at positions 9 and 18, and with lysine at position 14 since subunit 9 is coupled via lysine residues to the solid phase (Schmidt et al, 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. Two positive clones were obtained (inserts 107 and 108; length of the inserts ca.…”

The proton conducting F0-part of bacterial ATP synthases

“…The nucleotide sequence has been determined [14][15][16][17][18][19][20][21][22][23] and the sequence has been determined [81][82][83][84]. In yeast, subunit e (proteolipid subunit 9) is encoded on mitochondrial ONA [85], in other organisms in the nucleus [82][83][84][85][86]. The other genes have not been located, so far.…”

Biogenesis of Mitochondrial Energy Transducing Complexes

“…Furthermore, in many cases the content of serines and threonines is relatively high (Kaput et al, 1982;Viebrock et al, 1982;Maarse et al, 1984;Morohashi et al, 1984;Sadler et al, 1984;Sebald and Kruse, 1984;Suissa et al, 1984;Takiguchi et al, 1984;Wright et al, 1984;Gay and Walker, 1985;Guiard, 1985;Harnisch et al, 1985;Jaussi et al, 1985;Joh et al, 1985;Koerner et al, 1985;Marres et al, 1985;Mueckler and Pitot, 1985;Nyunoya et al, 1985;Takeda et al, 1985;Keng et al, 1986;Ohta and Kagawa, 1986). Subunit VI of the bc\ complex in yeast could be an exception, since it possesses a negatively charged amino-terminal sequence ; however, it is unknown where or whether at all it is proteolytically processed.…”

“…For example, subunit 9 of F 0 F r ATPase (proteolipid or dicyclohexylcarbodiimide (DCCD)-binding protein) from the fungus Neurospora crassa is a very hydrophobic protein of 81 amino acid residues. Its presequence is long (66 amino acid residues) and hydrophilic (Viebrock et al, 1982). Like most precursor proteins, subunit 9 is transported through the cytosol in the form of high-molecular-weight aggregates (Schmidt et al, 1983b).…”